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Early immune responses in rainbow trout liver upon viral hemorrhagic septicemia virus (VHSV) infection.

Castro R, Abós B, Pignatelli J, von Gersdorff Jørgensen L, González Granja A, Buchmann K, Tafalla C - PLoS ONE (2014)

Bottom Line: Our results indicate that T lymphocytes play a key role in the initial response to VHSV in the liver, since CD3, CD8, CD4, perforin, Mx and interferon (IFN) transcription levels were up-regulated in response to VHSV.Consequently, flow cytometry analysis of CD8α+ cells in liver and spleen at day 5 post-infection revealed a decrease in the number of CD8α+ cells in the spleen and an increased population in the liver.No differences were found however in the percentages of B lymphocyte (IgM+ or IgD+) populations.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación en Sanidad Animal (CISA-INIA), Valdeolmos (Madrid), Spain.

ABSTRACT
Among the essential metabolic functions of the liver, in mammals, a role as mediator of systemic and local innate immunity has also been reported. Although the presence of an important leukocyte population in mammalian liver is well documented, the characterization of leukocyte populations in the teleost liver has been only scarcely addressed. In the current work, we have confirmed the presence of IgM+, IgD+, IgT+, CD8α+, CD3+ cells, and cells expressing major histocompatibility complex (MHC-II) in rainbow trout (Oncorhynchus mykiss) liver by flow cytometry and/or immunohistochemistry analysis. Additionally, the effect of viral hemorrhagic septicemia virus (VHSV) on the liver immune response was assessed. First, we studied the effect of viral intraperitoneal injection on the transcription of a wide selection of immune genes at days 1, 2 and 5 post-infection. These included a group of leukocyte markers genes, pattern recognition receptors (PRRs), chemokines, chemokine receptor genes, and other genes involved in the early immune response and in acute phase reaction. Our results indicate that T lymphocytes play a key role in the initial response to VHSV in the liver, since CD3, CD8, CD4, perforin, Mx and interferon (IFN) transcription levels were up-regulated in response to VHSV. Consequently, flow cytometry analysis of CD8α+ cells in liver and spleen at day 5 post-infection revealed a decrease in the number of CD8α+ cells in the spleen and an increased population in the liver. No differences were found however in the percentages of B lymphocyte (IgM+ or IgD+) populations. In addition, a strong up-regulation in the transcription levels of several PRRs and chemokines was observed from the second day of infection, indicating an important role of these factors in the response of the liver to viral infections.

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Imunohistochemical detection of different leukocyte populations in trout liver.Representative photomicrographs of anti-IgM, anti-IgD, anti-IgT, anti-CD3, and anti-MHC-II positive staining in liver sections obtained from unstimulated fish (N = 4). Different phenotypes were observed among MHC-II+ cells including small lymphocyte-like round cells (arrows) and macrophage/dendritic-like cells (arrow heads). Some of these MHC-II+ cells appear as part of the endothelial layer of the blood vessels (asterisks). Counterstained with Mayer's haematoxylin. Scale bar represents 20 µm.
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pone-0111084-g001: Imunohistochemical detection of different leukocyte populations in trout liver.Representative photomicrographs of anti-IgM, anti-IgD, anti-IgT, anti-CD3, and anti-MHC-II positive staining in liver sections obtained from unstimulated fish (N = 4). Different phenotypes were observed among MHC-II+ cells including small lymphocyte-like round cells (arrows) and macrophage/dendritic-like cells (arrow heads). Some of these MHC-II+ cells appear as part of the endothelial layer of the blood vessels (asterisks). Counterstained with Mayer's haematoxylin. Scale bar represents 20 µm.

Mentions: Although the presence of IgM+ B cells in the liver was recently reported [13], [27], the presence of additional B and T cell populations has only been suggested. In the present work, we characterized the resident leukocyte population in unstimulated liver using mAb against specific leukocyte markers. The distribution of these leukocyte populations was studied by immunohistochemistry (IgM, IgD, IgT, MHC-II and CD3) and by flow cytometry (IgM, IgD, MHC-II and CD8α) using monoclonal antibodies previously characterized (Fig. S2) [21], [25], [28]. Through immunohistochemistry, as already reported [27], IgM+ cells were not only identified within the liver blood vessels but also homogeneously dispersed along the stroma of the liver, presenting a strong staining central core (Fig. 1). A similar distribution was observed for IgT+ cells. Some, less abundant, IgD+ and CD3+ cells were also observed dispersed along the parenchyma as small round cells with intense staining. MHC-II+ cells (that should mainly include B cells, dendritic cells and macrophages) are also homogeneously distributed along the liver, some of them with a lymphocyte-like morphology (small round cells with large nucleus), and other cells displaying a macrophage or dendritic-like morphology (big cells of irregular shape, with strong staining intensity in both the core of the cytoplasm and the prolongations of the cell). Interestingly, some of these MHC-II+ cells appear as part of the endothelial layer of the blood vessels (Fig. 1), in agreement with the antigen-presenting cell phenotype of mammalian hepatic endothelial cells, that includes the constitutive expression of MHC-II [29], [30].


Early immune responses in rainbow trout liver upon viral hemorrhagic septicemia virus (VHSV) infection.

Castro R, Abós B, Pignatelli J, von Gersdorff Jørgensen L, González Granja A, Buchmann K, Tafalla C - PLoS ONE (2014)

Imunohistochemical detection of different leukocyte populations in trout liver.Representative photomicrographs of anti-IgM, anti-IgD, anti-IgT, anti-CD3, and anti-MHC-II positive staining in liver sections obtained from unstimulated fish (N = 4). Different phenotypes were observed among MHC-II+ cells including small lymphocyte-like round cells (arrows) and macrophage/dendritic-like cells (arrow heads). Some of these MHC-II+ cells appear as part of the endothelial layer of the blood vessels (asterisks). Counterstained with Mayer's haematoxylin. Scale bar represents 20 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4206492&req=5

pone-0111084-g001: Imunohistochemical detection of different leukocyte populations in trout liver.Representative photomicrographs of anti-IgM, anti-IgD, anti-IgT, anti-CD3, and anti-MHC-II positive staining in liver sections obtained from unstimulated fish (N = 4). Different phenotypes were observed among MHC-II+ cells including small lymphocyte-like round cells (arrows) and macrophage/dendritic-like cells (arrow heads). Some of these MHC-II+ cells appear as part of the endothelial layer of the blood vessels (asterisks). Counterstained with Mayer's haematoxylin. Scale bar represents 20 µm.
Mentions: Although the presence of IgM+ B cells in the liver was recently reported [13], [27], the presence of additional B and T cell populations has only been suggested. In the present work, we characterized the resident leukocyte population in unstimulated liver using mAb against specific leukocyte markers. The distribution of these leukocyte populations was studied by immunohistochemistry (IgM, IgD, IgT, MHC-II and CD3) and by flow cytometry (IgM, IgD, MHC-II and CD8α) using monoclonal antibodies previously characterized (Fig. S2) [21], [25], [28]. Through immunohistochemistry, as already reported [27], IgM+ cells were not only identified within the liver blood vessels but also homogeneously dispersed along the stroma of the liver, presenting a strong staining central core (Fig. 1). A similar distribution was observed for IgT+ cells. Some, less abundant, IgD+ and CD3+ cells were also observed dispersed along the parenchyma as small round cells with intense staining. MHC-II+ cells (that should mainly include B cells, dendritic cells and macrophages) are also homogeneously distributed along the liver, some of them with a lymphocyte-like morphology (small round cells with large nucleus), and other cells displaying a macrophage or dendritic-like morphology (big cells of irregular shape, with strong staining intensity in both the core of the cytoplasm and the prolongations of the cell). Interestingly, some of these MHC-II+ cells appear as part of the endothelial layer of the blood vessels (Fig. 1), in agreement with the antigen-presenting cell phenotype of mammalian hepatic endothelial cells, that includes the constitutive expression of MHC-II [29], [30].

Bottom Line: Our results indicate that T lymphocytes play a key role in the initial response to VHSV in the liver, since CD3, CD8, CD4, perforin, Mx and interferon (IFN) transcription levels were up-regulated in response to VHSV.Consequently, flow cytometry analysis of CD8α+ cells in liver and spleen at day 5 post-infection revealed a decrease in the number of CD8α+ cells in the spleen and an increased population in the liver.No differences were found however in the percentages of B lymphocyte (IgM+ or IgD+) populations.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación en Sanidad Animal (CISA-INIA), Valdeolmos (Madrid), Spain.

ABSTRACT
Among the essential metabolic functions of the liver, in mammals, a role as mediator of systemic and local innate immunity has also been reported. Although the presence of an important leukocyte population in mammalian liver is well documented, the characterization of leukocyte populations in the teleost liver has been only scarcely addressed. In the current work, we have confirmed the presence of IgM+, IgD+, IgT+, CD8α+, CD3+ cells, and cells expressing major histocompatibility complex (MHC-II) in rainbow trout (Oncorhynchus mykiss) liver by flow cytometry and/or immunohistochemistry analysis. Additionally, the effect of viral hemorrhagic septicemia virus (VHSV) on the liver immune response was assessed. First, we studied the effect of viral intraperitoneal injection on the transcription of a wide selection of immune genes at days 1, 2 and 5 post-infection. These included a group of leukocyte markers genes, pattern recognition receptors (PRRs), chemokines, chemokine receptor genes, and other genes involved in the early immune response and in acute phase reaction. Our results indicate that T lymphocytes play a key role in the initial response to VHSV in the liver, since CD3, CD8, CD4, perforin, Mx and interferon (IFN) transcription levels were up-regulated in response to VHSV. Consequently, flow cytometry analysis of CD8α+ cells in liver and spleen at day 5 post-infection revealed a decrease in the number of CD8α+ cells in the spleen and an increased population in the liver. No differences were found however in the percentages of B lymphocyte (IgM+ or IgD+) populations. In addition, a strong up-regulation in the transcription levels of several PRRs and chemokines was observed from the second day of infection, indicating an important role of these factors in the response of the liver to viral infections.

Show MeSH
Related in: MedlinePlus