Intracellular bacteria interfere with dendritic cell functions: role of the type I interferon pathway.

Dendritic cells (DCs) orchestrate host defenses against microorganisms. In infectious diseases due to intracellular bacteria, the inefficiency of the immune system to eradicate microorganisms has been attributed to the hijacking of DC functions. In this study, we selected intracellular bacterial pathogens with distinct lifestyles and explored the responses of monocyte-derived DCs (moDCs). Using lipopolysaccharide as a control, we found that Orientia tsutsugamushi, the causative agent of scrub typhus that survives in the cytosol of target cells, induced moDC maturation, as assessed by decreased endocytosis activity, the ability to induce lymphocyte proliferation and the membrane expression of phenotypic markers. In contrast, Coxiella burnetii, the agent of Q fever, and Brucella abortus, the agent of brucellosis, both of which reside in vacuolar compartments, only partly induced the maturation of moDCs, as demonstrated by a phenotypic analysis. To analyze the mechanisms used by C. burnetii and B. abortus to alter moDC activation, we performed microarray and found that C. burnetii and B. abortus induced a specific signature consisting of TLR4, TLR3, STAT1 and interferon response genes. These genes were down-modulated in response to C. burnetii and B. abortus but up-modulated in moDCs activated by lipopolysaccharide and O. tsutsugamushi. This transcriptional alteration was associated with the defective interferon-β production. This study demonstrates that intracellular bacteria specifically affect moDC responses and emphasizes how C. burnetii and B. abortus interfere with moDC activation and the antimicrobial immune response. We believe that comparing infection by several bacterial species may be useful for defining new pathways and biomarkers and for developing new treatment strategies

Transcriptional program of stimulated moDCs.

<p>moDCs were stimulated with <i>B. abortus</i>, <i>C. burnetii</i>, <i>O. tsutsugamushi</i> or <i>E. coli</i> LPS for 6 hours. RNA was extracted, and a microarray was performed. <b>A</b>, A heatmap representation of the 3,610 modulated probes in stimulated moDCs compared to unstimulated moDCs (NS). The probes are shown in the rows and the samples in the columns. The expression levels are color coded from blue to red. <b>B</b>, Graphical representation of the samples based on the correspondence analysis of the modulated probes. The samples are colored according to the stimulus. <b>C</b>–<b>D</b>, Venn diagrams highlighting the core and specific signatures induced in moDCs stimulated with LPS and pathogens, showing up-modulated probes (C) and down-modulated probes (D). <b>E</b>, Summary of the pathways in moDCs modulated by intracellular bacteria. Ba: <i>B. abortus</i>; Cb: <i>C. burnetii</i>; Ot: <i>O. tsutsugamushi</i>.</p

Genes differentially expressed by bacterial pathogens.

<p>moDCs were stimulated with bacterial pathogens or <i>E. coli</i> LPS for 6 hours. RNAs were extracted, and microarrays were performed. The genes that were up-modulated by <i>O. tsutsugamushi</i> and LPS and down-modulated by <i>C. burnetii</i> and <i>B. abortus</i> are listed in alphabetic order. The values of FC are presented and IFN response genes are underlined.</p

KEGG pathways identified in stimulated moDCs.

<p>moDCs were stimulated with <i>E. coli</i> LPS or bacterial pathogens for 6 hours. RNAs were extracted, and microarrays were performed. The modulated genes were analyzed using the KEGG database. The KEGG pathways, the number of modulated genes and <i>P</i> values are shown.</p

Phenotypic study of moDC maturation.

<p>moDCs were stimulated with bacterial pathogens or <i>E. coli</i> LPS for 24 hours. The cells were then incubated with FITC-coupled anti-CD80 (A), PE-coupled anti-CD86 (B) and FITC-coupled anti-HLA-DR (C) Abs for 30 minutes and analyzed by flow cytometry. The histograms are representative of three different experiments. The bar charts represent the MFI FC values of stimulated moDCs compared with unstimulated moDCs. *p</p

Functional study of moDC maturation.

<p><b>A</b>, moDCs were stimulated with <i>T. whipplei</i> (a), <i>B. abortus</i> (b), <i>C. burnetii</i> (c), <i>O. tsutsugamushi</i> (d) or <i>E. coli</i> LPS (e) for 24 hours and then incubated with FITC-albumin (1 mg/mL) for 1 hour. The fluorescence intensities of unstimulated moDCs and stimulated moDCs were determined by flow cytometry. The results are expressed as the ratio of stimulated moDC fluorescence over unstimulated moDC fluorescence (f). They represent the mean ± SD of three independent experiments. ***p<0.001 represents the comparison of stimulated moDCs vs unstimulated DCs using Student's t-test. <b>B</b>, moDCs stimulated with <i>T. whipplei</i> (a), <i>B. abortus</i> (b), <i>C. burnetii</i> (c), <i>O. tsutsugamushi</i> (d) or <i>E. coli</i> LPS (e) were co-cultured with CFSE-labeled autologous T-lymphocytes for 5 days. The gating strategy used to determine lymphocyte proliferation is shown for each stimulation (a–e). The results are expressed as the percentage of new T-cells (cells with decreased fluorescence intensity) in the total T-cell population (f). They represent the mean ± SD of three independent experiments. **p<0.02 represents the comparison of stimulated moDCs vs unstimulated DCs using Student's t-test. <b>C</b>, The membrane expression of CD83 by moDCs incubated with bacterial pathogens or LPS for 24 hours was determined by flow cytometry using PE-coupled anti-CD83 Abs (a). The histograms are representative of three different experiments. The FC of MFI values after stimulation was compared to unstimulated condition (b). *p<0.03 represents the comparison of stimulated moDCs vs unstimulated DCs using Student's t-test.</p

The ARCHES Space-Analogue Demonstration Mission: Towards Heterogeneous Teams of Autonomous Robots for Collaborative Scientific Sampling in Planetary Exploration

Teams of mobile robots will play a crucial role in future missions to explore the surfaces of extraterrestrial bodies. Setting up infrastructure and taking scientific samples are expensive tasks when operating in distant, challenging, and unknown environments. In contrast to current single-robot space missions, future heterogeneous robotic teams will increase efficiency via enhanced autonomy and parallelization, improve robustness via functional redundancy, as well as benefit from complementary capabilities of the individual robots. In this letter, we present our heterogeneous robotic team, consisting of flying and driving robots that we plan to deploy on scientific sampling demonstration missions at a Moon-analogue site on Mt. Etna, Sicily, Italy in 2021 as part of the ARCHES project. We describe the robots' individual capabilities and their roles in two mission scenarios. We then present components and experiments on important tasks therein: automated task planning, high-level mission control, spectral rock analysis, radio-based localization, collaborative multi-robot 6D SLAM in Moon-analogue and Mars-like scenarios, and demonstrations of autonomous sample return

Brucella pinnipedialis in hooded seal (Cystophora cristata) primary epithelial cells

Background: Marine Brucella spp. have been isolated from numerous pinniped and cetacean species, but pathological findings in association with infection with Brucella pinnipedialis in pinnipeds have been sparse. The capacity of brucellae to survive and replicate within host macrophages underlies their important ability to produce chronic infections, but previous work has shown that B. pinnipedialis spp. are rapidly eliminated from hooded seal (Cystophora cristata) alveolar macrophages. Results: To investigate if multiplication could take place in other hooded seal cell types, primary epithelial cells were isolated, verified to express the epithelial marker cytokeratin and challenged with three different strains of B. pinnipedialis; B. pinnipedialis sp. nov., B. pinnipedialis hooded seal strain B17, and B. pinnipedialis hooded seal strain 22F1. All strains were steadily eliminated and the amounts of intracellular bacteria were reduced to less than one-third by 48 h post infection. Intracellular presence was verified using immunocytochemistry. Conclusions: So far, intracellular multiplication in seal cells has not been documented for B. pinnipedialis. The lack of intracellular survival in macrophages, as well as in epithelial cells, together with the fact that pathological changes due to B. pinnipedialis infection is not yet identified in seals, suggests that the bacteria may only cause a mild, acute and transient infection. These findings also contribute to substantiate the hypothesis that seals may not be the primary host of B. pinnipedialis and that the transmission to seals are caused by other species in the marine environment

The ARCHES Moon-Analogue Demonstration Mission: Towards Teams of Autonomous Robots for Collaborative Scientific Sampling in Lunar Environments

Teams of mobile robots will play a crucial role in future missions to explore the surface of the Moon. Setting up infrastructure and taking scientific samples are expensive tasks when operating in such distant, challenging, and unknown environments. In contrast to current single-robot space missions, future heterogeneous robotic teams will increase efficiency via enhanced autonomy and parallelization, improve robustness via functional redundancy, as well as benefit from complementary capabilities of the individual robots. We present our heterogeneous robotic team, consisting of flying and driving robots that we plan to deploy on scientific sampling demonstration missions at a Moon-analogue site on Mt. Etna, Sicily, Italy in 2021 as part of the ARCHES project. We give a brief description of the robots' complementary capabilities and present their roles in two mission scenarios