In Situ Microscopy Analysis Reveals Local Innate Immune Response Developed around Brucella Infected Cells in Resistant and Susceptible Mice

Brucella are facultative intracellular bacteria that chronically infect humans and animals causing brucellosis. Brucella are able to invade and replicate in a broad range of cell lines in vitro, however the cells supporting bacterial growth in vivo are largely unknown. In order to identify these, we used a Brucella melitensis strain stably expressing mCherry fluorescent protein to determine the phenotype of infected cells in spleen and liver, two major sites of B. melitensis growth in mice. In both tissues, the majority of primary infected cells expressed the F4/80 myeloid marker. The peak of infection correlated with granuloma development. These structures were mainly composed of CD11b+ F4/80+ MHC-II+ cells expressing iNOS/NOS2 enzyme. A fraction of these cells also expressed CD11c marker and appeared similar to inflammatory dendritic cells (DCs). Analysis of genetically deficient mice revealed that differentiation of iNOS+ inflammatory DC, granuloma formation and control of bacterial growth were deeply affected by the absence of MyD88, IL-12p35 and IFN-γ molecules. During chronic phase of infection in susceptible mice, we identified a particular subset of DC expressing both CD11c and CD205, serving as a reservoir for the bacteria. Taken together, our results describe the cellular nature of immune effectors involved during Brucella infection and reveal a previously unappreciated role for DC subsets, both as effectors and reservoir cells, in the pathogenesis of brucellosis

Identification of immune effectors essential to the control of primary and secondary intranasal infection with brucella melitensis in mice

The mucosal immune system represents the first line of defense against Brucella infection in nature. We used genetically deficient mice to identify the lymphocytes and signaling pathways implicated in the control of primary and secondary intranasal infection with B. melitensis. Our analysis of primary infection demonstrated that the effectors implicated differ at the early and late stages and are dependent on the organ. TCR-d, TAP1, and IL-17RA deficiency specifically affects early control of Brucella in the lungs, whereas MHC class II (MHCII) and IFN-γR deficiency impairs late control in the lungs, spleen, and liver. Interestingly, IL-12p352/2 mice display enhanced Brucella growth in the spleen but not in the lungs or liver. Secondary intranasal infections are efficiently contained in the lung. In contrast to an i.p. infectious model, in which IL-12p35, MHCII, and B cells are strictly required for the control of secondary infection, we observed that only TCR-b deficiency or simultaneous neutralization of IL-12p35-and IL-17A-dependent pathways impairs the memory protective response against a secondary intranasal infection. Protection is not affected by TCR-d, MHCII, TAP1, B cell, IL-17RA, or IL-12p35 deficiency, suggesting that CD4+ and CD8+ a/b+ T cells are sufficient to mount a protective immune response and that an IL-17A-mediated response can compensate for the partial deficiency of an IFN-γ-mediated response to control a Brucella challenge. These findings demonstrate that the nature of the protective memory response depends closely on the route of infection and highlights the role of IFN-γ-and IL-17RA-mediated responses in the control of mucosal infection by Brucella.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Gateway to genetic exchange? DNA double-strand breaks in the bdelloid rotifer Adineta vaga submitted to desiccation.

International audienceThe bdelloid rotifer lineage Adineta vaga inhabits temporary habitats subjected to frequent episodes of drought. The recently published draft sequence of the genome of A. vaga revealed a peculiar genomic structure incompatible with meiosis and suggesting that DNA damage induced by desiccation may have reshaped the genomic structure of these organisms. However, the causative link between DNA damage and desiccation has never been proven to date in rotifers. To test for the hypothesis that desiccation induces DNA double-strand breaks (DSBs), we developed a protocol allowing a high survival rate of desiccated A. vaga. Using pulsed-field gel electrophoresis to monitor genomic integrity, we followed the occurrence of DSBs in dried bdelloids and observed an accumulation of these breaks with time spent in dehydrated state. These DSBs are gradually repaired upon rehydration. Even when the genome was entirely shattered into small DNA fragments by proton radiation, A. vaga individuals were able to efficiently recover from desiccation and repair a large amount of DSBs. Interestingly, when investigating the influence of UV-A and UV-B exposure on the genomic integrity of desiccated bdelloids, we observed that these natural radiations also caused important DNA DSBs, suggesting that the genome is not protected during the desiccated stage but that the repair mechanisms are extremely efficient in these intriguing organisms

Course of <i>B</i>. <i>melitensis</i> infection in organs of wild-type (wt), STAT6-, IL12p40- and STAT6/IL12p40-deficient BALB/c mice.

<p>The mice were injected intranasally (i.n.) with 2x10⁷ CFU of mCherry-Br <i>B</i>. <i>melitensis</i> and sacrificed at the indicated times. The data represent the number of CFU per gram of lung, spleen and liver. Grey bars represent the medians. “n” is the number of mice used. These results are representative of at least two independent experiments. ns, non-significant.</p

Characterization of the cell surface phenotype of infected cells in the spleen of IL12p40-deficient BALB/c mice.

<p>IL12p40^-/- STAT6^+/+ BALB/c mice were injected i.n. with 2x10⁷ CFU of mCherry-Br. The mice were sacrificed at 28 days post-infection and the spleens were collected and examined by immunohistofluorescence. <b>A</b>. The left panels show the overall distribution of the CD11c-, ERTR9 and MOMA-1-expressing cells in the spleen. The panels to the right of the first ones show mCherry-Br co-localization with negative cells and positive cells for CD11c, ER-TR9 and MOMA-1. The panels are color-coded with the text for phalloidin, the antigen examined or mCherry-Br. Scale bar = 200 and 20 μm, as indicated. r.p.: red pulp; w.p.: white pulp. Yellow arrowheads indicate the presence of bacteria. The data are representative of at least three independent experiments. <b>B</b>. Representative confocal images of mCherry-Br infected CD11c⁺ cells. The panels are color-coded with the text for DAPI, mCherry-Br or CD11c. Scale bar = 10 μm, as indicated. w.p.: white pulp.</p

Characterization of infected cells expressing CD11c, CD205 and MOMA-1 in the spleen of IL12p40-deficient BALB/c mice.

<p>IL12p40^-/- STAT6^+/+ BALB/c mice were injected i.n. with 2x10⁷ CFU of mCherry-Br. The mice were sacrificed at 28 days post-infection and the spleens were collected and examined by immunohistofluorescence. <b>A</b>, The left panel shows mCherry-Br co-localization with cells expressing CD11c. The middle panel shows mCherry-Br co-localization with cells expressing CD205 and the right panel shows co-localization of CD11c- and CD205-expressing cells. <b>B</b>, The upper panels show distribution of MOMA-1-expressing cells and co-localization with mCherry-Br (left), distribution of DEC205-expressing cells and co-localization with mCherry-Br (middle), and co-localization of MOMA-1- and CD205-expressing cells (right). The panels below are higher magnification views of the same stainings. The panels are color-coded with the text for phalloidin, the antigen examined or mCherry-Br. Scale bar = 50 and 20 μm, as indicated. r.p.: red pulp; w.p.: white pulp. Yellow arrowheads indicate the presence of bacteria. The data are representative of at least two independent experiments.</p

Imported human brucellosis in Belgium: Bio and molecular typing of bacterial isolates, 1996-2015

The aim of this study was to characterize by classical biotyping and Multi-Locus variable number tandem repeats (VNTR) Analysis (MLVA) all Brucella spp. derived from human cases in Belgium from 1996 to 2015. Final goals were to determine the species and biovar, to trace-back on genetic grounds the origin of each strain when patient history and risk factors were missing, and to survey for particular trends at the national leve