Video_2_Platelets Promote Brucella abortus Monocyte Invasion by Establishing Complexes With Monocytes.AVI

<p>Brucellosis is an infectious disease elicited by bacteria of the genus Brucella. Platelets have been extensively described as mediators of hemostasis and responsible for maintaining vascular integrity. Nevertheless, they have been recently involved in the modulation of innate and adaptive immune responses. Although many interactions have been described between Brucella abortus and monocytes/macrophages, the role of platelets during monocyte/macrophage infection by these bacteria remained unknown. The aim of this study was to investigate the role of platelets in the immune response against B. abortus. We first focused on the possible interactions between B. abortus and platelets. Bacteria were able to directly interact with platelets. Moreover, this interaction triggered platelet activation, measured as fibrinogen binding and P-selectin expression. We further investigated whether platelets were involved in Brucella-mediated monocyte/macrophage early infection. The presence of platelets promoted the invasion of monocytes/macrophages by B. abortus. Moreover, platelets established complexes with infected monocytes/macrophages as a result of a carrier function elicited by platelets. We also evaluated the ability of platelets to modulate functional aspects of monocytes in the context of the infection. The presence of platelets during monocyte infection enhanced IL-1β, TNF-α, IL-8, and MCP-1 secretion while it inhibited the secretion of IL-10. At the same time, platelets increased the expression of CD54 (ICAM-1) and CD40. Furthermore, we showed that soluble factors released by B. abortus-activated platelets, such as soluble CD40L, platelet factor 4, platelet-activating factor, and thromboxane A₂, were involved in CD54 induction. Overall, our results indicate that platelets can directly sense and react to B. abortus presence and modulate B. abortus-mediated infection of monocytes/macrophages increasing their pro-inflammatory capacity, which could promote the resolution of the infection.</p

Image_1_Platelets Promote Brucella abortus Monocyte Invasion by Establishing Complexes With Monocytes.TIF

<p>Brucellosis is an infectious disease elicited by bacteria of the genus Brucella. Platelets have been extensively described as mediators of hemostasis and responsible for maintaining vascular integrity. Nevertheless, they have been recently involved in the modulation of innate and adaptive immune responses. Although many interactions have been described between Brucella abortus and monocytes/macrophages, the role of platelets during monocyte/macrophage infection by these bacteria remained unknown. The aim of this study was to investigate the role of platelets in the immune response against B. abortus. We first focused on the possible interactions between B. abortus and platelets. Bacteria were able to directly interact with platelets. Moreover, this interaction triggered platelet activation, measured as fibrinogen binding and P-selectin expression. We further investigated whether platelets were involved in Brucella-mediated monocyte/macrophage early infection. The presence of platelets promoted the invasion of monocytes/macrophages by B. abortus. Moreover, platelets established complexes with infected monocytes/macrophages as a result of a carrier function elicited by platelets. We also evaluated the ability of platelets to modulate functional aspects of monocytes in the context of the infection. The presence of platelets during monocyte infection enhanced IL-1β, TNF-α, IL-8, and MCP-1 secretion while it inhibited the secretion of IL-10. At the same time, platelets increased the expression of CD54 (ICAM-1) and CD40. Furthermore, we showed that soluble factors released by B. abortus-activated platelets, such as soluble CD40L, platelet factor 4, platelet-activating factor, and thromboxane A₂, were involved in CD54 induction. Overall, our results indicate that platelets can directly sense and react to B. abortus presence and modulate B. abortus-mediated infection of monocytes/macrophages increasing their pro-inflammatory capacity, which could promote the resolution of the infection.</p

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

<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

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-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