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In Situ Characterization of Splenic Brucella melitensis Reservoir Cells during the Chronic Phase of Infection in Susceptible Mice.

Hanot Mambres D, Machelart A, Vanderwinden JM, De Trez C, Ryffel B, Letesson JJ, Muraille E - PLoS ONE (2015)

Bottom Line: We performed an in situ analysis by fluorescent microscopy of the phenotype of B. melitensis infected spleen cells from intranasally infected IL-12p40-/- BALB/c mice and the impact of STAT6 deficiency on this phenotype.Most of the infected spleen cells contained high levels of lipids and expressed CD11c and CD205 dendritic cell markers and Arginase1, but were negative for the M2a markers Fizz1 or CD301.This characterization of B. melitensis reservoir cells could provide a better understanding of Brucella persistence in the host and lead to the design of more efficient therapeutic strategies.

View Article: PubMed Central - PubMed

Affiliation: Unité de Recherche en Biologie des Microorganismes, Laboratoire d'Immunologie et de Microbiologie, Université de Namur, Namur, Belgium.

ABSTRACT
Brucella are facultative intracellular Gram-negative coccobacilli that chronically infect humans as well as domestic and wild-type mammals, and cause brucellosis. Alternatively activated macrophages (M2a) induced by IL-4/IL-13 via STAT6 signaling pathways have been frequently described as a favorable niche for long-term persistence of intracellular pathogens. Based on the observation that M2a-like macrophages are induced in the spleen during the chronic phase of B. abortus infection in mice and are strongly infected in vitro, it has been suggested that M2a macrophages could be a potential in vivo niche for Brucella. In order to test this hypothesis, we used a model in which infected cells can be observed directly in situ and where the differentiation of M2a macrophages is favored by the absence of an IL-12-dependent Th1 response. We performed an in situ analysis by fluorescent microscopy of the phenotype of B. melitensis infected spleen cells from intranasally infected IL-12p40-/- BALB/c mice and the impact of STAT6 deficiency on this phenotype. Most of the infected spleen cells contained high levels of lipids and expressed CD11c and CD205 dendritic cell markers and Arginase1, but were negative for the M2a markers Fizz1 or CD301. Furthermore, STAT6 deficiency had no effect on bacterial growth or the reservoir cell phenotype in vivo, leading us to conclude that, in our model, the infected cells were not Th2-induced M2a macrophages. This characterization of B. melitensis reservoir cells could provide a better understanding of Brucella persistence in the host and lead to the design of more efficient therapeutic strategies.

No MeSH data available.


Related in: MedlinePlus

Characterization of the cell surface phenotype of infected cells in the spleen of IL12p40-deficient BALB/c mice.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. A. 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. 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.
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pone.0137835.g003: Characterization of the cell surface phenotype of infected cells in the spleen of IL12p40-deficient BALB/c mice.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. A. 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. 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.

Mentions: In order to determine the impact of STAT6 deficiency on the phenotype of infected spleen cells, we compared in situ using fluorescence microscopy the expression of various cell surface markers on infected cells in the spleen from IL-12p40-/- and IL-12p40-/- STAT6-/- BALB/c mice at 28 days post-infection. We analyzed the expression of GR1, F4/80, CD11b, CD11c, CD8α, MHC-II, MOMA-1 and ER-TR9 markers on a minimum of 200 infected cells from 3 animals per group. This analysis was performed under blinded conditions to reduce the risk of biased results, and it was repeated in two distinct experimental infections. The frequency of each negative and positive infected cells from both mouse strains is presented in Fig 2. No significant difference was observed between the IL-12p40-/- STAT6+/+ and IL-12p40-/- STAT6-/- mice, whatever the markers tested. In consequence, we only present images from IL-12p40-/- STAT6+/+ mice to illustrate each staining. The general distribution for each staining and an example of negative and positive cells is presented in Fig 3A and S2 Fig. In order to further confirm the expression of CD11c, MOMA-1 and ER-TR9 by infected cells, a confocal analysis was also performed on the spleen section (Fig 3B for CD11c, data not shown for MOMA-1 and ER-TR9).


In Situ Characterization of Splenic Brucella melitensis Reservoir Cells during the Chronic Phase of Infection in Susceptible Mice.

Hanot Mambres D, Machelart A, Vanderwinden JM, De Trez C, Ryffel B, Letesson JJ, Muraille E - PLoS ONE (2015)

Characterization of the cell surface phenotype of infected cells in the spleen of IL12p40-deficient BALB/c mice.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. A. 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. 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.
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pone.0137835.g003: Characterization of the cell surface phenotype of infected cells in the spleen of IL12p40-deficient BALB/c mice.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. A. 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. 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.
Mentions: In order to determine the impact of STAT6 deficiency on the phenotype of infected spleen cells, we compared in situ using fluorescence microscopy the expression of various cell surface markers on infected cells in the spleen from IL-12p40-/- and IL-12p40-/- STAT6-/- BALB/c mice at 28 days post-infection. We analyzed the expression of GR1, F4/80, CD11b, CD11c, CD8α, MHC-II, MOMA-1 and ER-TR9 markers on a minimum of 200 infected cells from 3 animals per group. This analysis was performed under blinded conditions to reduce the risk of biased results, and it was repeated in two distinct experimental infections. The frequency of each negative and positive infected cells from both mouse strains is presented in Fig 2. No significant difference was observed between the IL-12p40-/- STAT6+/+ and IL-12p40-/- STAT6-/- mice, whatever the markers tested. In consequence, we only present images from IL-12p40-/- STAT6+/+ mice to illustrate each staining. The general distribution for each staining and an example of negative and positive cells is presented in Fig 3A and S2 Fig. In order to further confirm the expression of CD11c, MOMA-1 and ER-TR9 by infected cells, a confocal analysis was also performed on the spleen section (Fig 3B for CD11c, data not shown for MOMA-1 and ER-TR9).

Bottom Line: We performed an in situ analysis by fluorescent microscopy of the phenotype of B. melitensis infected spleen cells from intranasally infected IL-12p40-/- BALB/c mice and the impact of STAT6 deficiency on this phenotype.Most of the infected spleen cells contained high levels of lipids and expressed CD11c and CD205 dendritic cell markers and Arginase1, but were negative for the M2a markers Fizz1 or CD301.This characterization of B. melitensis reservoir cells could provide a better understanding of Brucella persistence in the host and lead to the design of more efficient therapeutic strategies.

View Article: PubMed Central - PubMed

Affiliation: Unité de Recherche en Biologie des Microorganismes, Laboratoire d'Immunologie et de Microbiologie, Université de Namur, Namur, Belgium.

ABSTRACT
Brucella are facultative intracellular Gram-negative coccobacilli that chronically infect humans as well as domestic and wild-type mammals, and cause brucellosis. Alternatively activated macrophages (M2a) induced by IL-4/IL-13 via STAT6 signaling pathways have been frequently described as a favorable niche for long-term persistence of intracellular pathogens. Based on the observation that M2a-like macrophages are induced in the spleen during the chronic phase of B. abortus infection in mice and are strongly infected in vitro, it has been suggested that M2a macrophages could be a potential in vivo niche for Brucella. In order to test this hypothesis, we used a model in which infected cells can be observed directly in situ and where the differentiation of M2a macrophages is favored by the absence of an IL-12-dependent Th1 response. We performed an in situ analysis by fluorescent microscopy of the phenotype of B. melitensis infected spleen cells from intranasally infected IL-12p40-/- BALB/c mice and the impact of STAT6 deficiency on this phenotype. Most of the infected spleen cells contained high levels of lipids and expressed CD11c and CD205 dendritic cell markers and Arginase1, but were negative for the M2a markers Fizz1 or CD301. Furthermore, STAT6 deficiency had no effect on bacterial growth or the reservoir cell phenotype in vivo, leading us to conclude that, in our model, the infected cells were not Th2-induced M2a macrophages. This characterization of B. melitensis reservoir cells could provide a better understanding of Brucella persistence in the host and lead to the design of more efficient therapeutic strategies.

No MeSH data available.


Related in: MedlinePlus