Limits...
Jaagsiekte sheep retrovirus infection of lung slice cultures.

Cousens C, Alleaume C, Bijsmans E, Martineau HM, Finlayson J, Dagleish MP, Griffiths DJ - Retrovirology (2015)

Bottom Line: Interestingly, the JSRV Env protein is directly oncogenic and capable of driving cellular transformation in vivo and in vitro.JSRV was able to infect cells within lung slices, to produce new infectious virions and induce cell proliferation.Immunohistochemical labeling revealed that infected lung slice cells express markers of type II pneumocytes and phosphorylated Akt and ERK1/2.

View Article: PubMed Central - PubMed

Affiliation: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Edinburgh, UK. chris.cousens@moredun.ac.uk.

ABSTRACT

Background: Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA), a transmissible neoplastic disease of sheep. OPA is an economically important veterinary disease and is also a valuable naturally occurring animal model of human lung cancer, with which it shares a similar histological appearance and the activation of common cell signaling pathways. Interestingly, the JSRV Env protein is directly oncogenic and capable of driving cellular transformation in vivo and in vitro. Previous studies of JSRV infection in cell culture have been hindered by the lack of a permissive cell line for the virus. Here, we investigated the ability of JSRV to infect slices of ovine lung tissue cultured ex vivo.

Results: We describe the use of precision cut lung slices from healthy sheep to study JSRV infection and transformation ex vivo. Following optimization of the culture system we characterized JSRV infection of lung slices and compared the phenotype of infected cells to natural field cases and to experimentally-induced OPA tumors from sheep. JSRV was able to infect cells within lung slices, to produce new infectious virions and induce cell proliferation. Immunohistochemical labeling revealed that infected lung slice cells express markers of type II pneumocytes and phosphorylated Akt and ERK1/2. These features closely resemble the phenotype of natural and experimentally-derived OPA in sheep, indicating that lung slice culture provides an authentic ex vivo model of OPA.

Conclusions: We conclude that we have established an ex vivo model of JSRV infection. This model will be valuable for future studies of JSRV replication and early events in oncogenesis and provides a novel platform for studies of JSRV-induced lung cancer.

Show MeSH

Related in: MedlinePlus

Viability and structure of cultured ovine lung slices. Hematoxylin and eosin (HE) stained uninfected lung slices at 0 (A), 16 (B) and 42 (C) dpi. In panel B the arrows indicate areas of “thickening” around the edges of the lung slice. In panel C the small arrow indicates epithelial cells and the large arrow indicates necrotic interstitial cells. D, E, F) IHC of d16 lung slices labeled with anti-Ki-67 (D), anti-pan-cytokeratin (E), and anti-DC-LAMP (F). Lung slices D, E and F were treated with the replication-defective JSRV-∆RT virus.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4419405&req=5

Fig1: Viability and structure of cultured ovine lung slices. Hematoxylin and eosin (HE) stained uninfected lung slices at 0 (A), 16 (B) and 42 (C) dpi. In panel B the arrows indicate areas of “thickening” around the edges of the lung slice. In panel C the small arrow indicates epithelial cells and the large arrow indicates necrotic interstitial cells. D, E, F) IHC of d16 lung slices labeled with anti-Ki-67 (D), anti-pan-cytokeratin (E), and anti-DC-LAMP (F). Lung slices D, E and F were treated with the replication-defective JSRV-∆RT virus.

Mentions: Morphological changes due to hyper-cellularity were noticeable from around day 8 in culture, and were particularly marked around the cut edges of the slices (Figure 1A, B). Similar gross changes were seen both in JSRV-infected or uninfected lung slices demonstrating that the aberrant growth was not due to JSRV infection but instead appears to be a reaction of the tissue to processing and/or in vitro culture. After an extended time in culture (42 days) the epithelial cells continued to appear histologically normal whereas interstitial cells appeared degenerate (Figure 1C). IHC labeling with an antibody to the proliferation marker Ki-67 suggested that the structural changes in the first weeks of culture were due to proliferation of cells in the interstitial compartment (Figure 1D). There was also an increase in cuboidal cells lining the alveoli which were positive for pan-cytokeratin (an epithelial cell marker) (Figure 1E) and DC-LAMP (type II pneumocytes) (Figure 1F), indicative of type II pneumocyte hyperplasia, which may be a response of the tissue to injury caused by slicing. This analysis indicated that ovine lung slices can survive in culture for at least 6 weeks whilst retaining many of the characteristics of normal lung tissue.Figure 1


Jaagsiekte sheep retrovirus infection of lung slice cultures.

Cousens C, Alleaume C, Bijsmans E, Martineau HM, Finlayson J, Dagleish MP, Griffiths DJ - Retrovirology (2015)

Viability and structure of cultured ovine lung slices. Hematoxylin and eosin (HE) stained uninfected lung slices at 0 (A), 16 (B) and 42 (C) dpi. In panel B the arrows indicate areas of “thickening” around the edges of the lung slice. In panel C the small arrow indicates epithelial cells and the large arrow indicates necrotic interstitial cells. D, E, F) IHC of d16 lung slices labeled with anti-Ki-67 (D), anti-pan-cytokeratin (E), and anti-DC-LAMP (F). Lung slices D, E and F were treated with the replication-defective JSRV-∆RT virus.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4419405&req=5

Fig1: Viability and structure of cultured ovine lung slices. Hematoxylin and eosin (HE) stained uninfected lung slices at 0 (A), 16 (B) and 42 (C) dpi. In panel B the arrows indicate areas of “thickening” around the edges of the lung slice. In panel C the small arrow indicates epithelial cells and the large arrow indicates necrotic interstitial cells. D, E, F) IHC of d16 lung slices labeled with anti-Ki-67 (D), anti-pan-cytokeratin (E), and anti-DC-LAMP (F). Lung slices D, E and F were treated with the replication-defective JSRV-∆RT virus.
Mentions: Morphological changes due to hyper-cellularity were noticeable from around day 8 in culture, and were particularly marked around the cut edges of the slices (Figure 1A, B). Similar gross changes were seen both in JSRV-infected or uninfected lung slices demonstrating that the aberrant growth was not due to JSRV infection but instead appears to be a reaction of the tissue to processing and/or in vitro culture. After an extended time in culture (42 days) the epithelial cells continued to appear histologically normal whereas interstitial cells appeared degenerate (Figure 1C). IHC labeling with an antibody to the proliferation marker Ki-67 suggested that the structural changes in the first weeks of culture were due to proliferation of cells in the interstitial compartment (Figure 1D). There was also an increase in cuboidal cells lining the alveoli which were positive for pan-cytokeratin (an epithelial cell marker) (Figure 1E) and DC-LAMP (type II pneumocytes) (Figure 1F), indicative of type II pneumocyte hyperplasia, which may be a response of the tissue to injury caused by slicing. This analysis indicated that ovine lung slices can survive in culture for at least 6 weeks whilst retaining many of the characteristics of normal lung tissue.Figure 1

Bottom Line: Interestingly, the JSRV Env protein is directly oncogenic and capable of driving cellular transformation in vivo and in vitro.JSRV was able to infect cells within lung slices, to produce new infectious virions and induce cell proliferation.Immunohistochemical labeling revealed that infected lung slice cells express markers of type II pneumocytes and phosphorylated Akt and ERK1/2.

View Article: PubMed Central - PubMed

Affiliation: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Edinburgh, UK. chris.cousens@moredun.ac.uk.

ABSTRACT

Background: Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma (OPA), a transmissible neoplastic disease of sheep. OPA is an economically important veterinary disease and is also a valuable naturally occurring animal model of human lung cancer, with which it shares a similar histological appearance and the activation of common cell signaling pathways. Interestingly, the JSRV Env protein is directly oncogenic and capable of driving cellular transformation in vivo and in vitro. Previous studies of JSRV infection in cell culture have been hindered by the lack of a permissive cell line for the virus. Here, we investigated the ability of JSRV to infect slices of ovine lung tissue cultured ex vivo.

Results: We describe the use of precision cut lung slices from healthy sheep to study JSRV infection and transformation ex vivo. Following optimization of the culture system we characterized JSRV infection of lung slices and compared the phenotype of infected cells to natural field cases and to experimentally-induced OPA tumors from sheep. JSRV was able to infect cells within lung slices, to produce new infectious virions and induce cell proliferation. Immunohistochemical labeling revealed that infected lung slice cells express markers of type II pneumocytes and phosphorylated Akt and ERK1/2. These features closely resemble the phenotype of natural and experimentally-derived OPA in sheep, indicating that lung slice culture provides an authentic ex vivo model of OPA.

Conclusions: We conclude that we have established an ex vivo model of JSRV infection. This model will be valuable for future studies of JSRV replication and early events in oncogenesis and provides a novel platform for studies of JSRV-induced lung cancer.

Show MeSH
Related in: MedlinePlus