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Phenotypic and functional characterization of the major lymphocyte populations in the fruit-eating bat Pteropus alecto

View Article: PubMed Central - PubMed

ABSTRACT

The unique ability of bats to act as reservoir for viruses that are highly pathogenic to humans suggests unique properties and functional characteristics of their immune system. However, the lack of bat specific reagents, in particular antibodies, has limited our knowledge of bat’s immunity. Using cross-reactive antibodies, we report the phenotypic and functional characterization of T cell subsets, B and NK cells in the fruit-eating bat Pteropus alecto. Our findings indicate the predominance of CD8+ T cells in the spleen from wild-caught bats that may reflect either the presence of viruses in this organ or predominance of this cell subset at steady state. Instead majority of T cells in circulation, lymph nodes and bone marrow (BM) were CD4+ subsets. Interestingly, 40% of spleen T cells expressed constitutively IL-17, IL-22 and TGF-β mRNA, which may indicate a strong bias towards the Th17 and regulatory T cell subsets. Furthermore, the unexpected high number of T cells in bats BM could suggest an important role in T cell development. Finally, mitogenic stimulation induced proliferation and production of effector molecules by bats immune cells. This work contributes to a better understanding of bat’s immunity, opening up new perspectives of therapeutic interventions for humans.

No MeSH data available.


Bat T cell production of cytokines and cytolytic factors upon mitogenic stimulation.Bat splenocytes (a–c,g–i) or PBMCs (d–f) were stimulated for 4 h with PDBu/ionomycin or media in the presence of brefeldin A and monensin. Detection of intracellular TNF (a), IFN-γ (b), IL-10 (c) was performed at the protein level, and production of IL- 2 (d), granzyme B (e), perforin (f), IL-17a (g), IL-22 (h) and TGF-β1 (i) was detected at the mRNA level by Flow-FISH. Dot plots obtained with one bat are shown and are representative of the results obtained with 2–3 different bats.
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f5: Bat T cell production of cytokines and cytolytic factors upon mitogenic stimulation.Bat splenocytes (a–c,g–i) or PBMCs (d–f) were stimulated for 4 h with PDBu/ionomycin or media in the presence of brefeldin A and monensin. Detection of intracellular TNF (a), IFN-γ (b), IL-10 (c) was performed at the protein level, and production of IL- 2 (d), granzyme B (e), perforin (f), IL-17a (g), IL-22 (h) and TGF-β1 (i) was detected at the mRNA level by Flow-FISH. Dot plots obtained with one bat are shown and are representative of the results obtained with 2–3 different bats.

Mentions: To gain further insights into the functionality of P. alecto immune system, an ex-vivo mitogenic stimulation experiment was performed. Bat splenocytes or PBMCs were stimulated with PDBu and the ionophore ionomycin (activation of protein kinase C bypassing the T cell receptor), in the presence of brefeldin A and monensin that block the Golgi apparatus and allow intracellular accumulation of cytokines produced in response to the stimulation. Cross-reactive anti-human TNF and anti-human IL-10 antibodies, as well as anti-bat IFN-γ antibody were used to detect by flow cytometry the production of these cytokines. In addition, Flow-FISH was used to detect the production at the mRNA level of IL-2 (T cell growth factor), granzyme B and perforin (cytolytic molecules mainly produced by CD8+ T cells and NK cells103738), IL-17A and IL-22 (inflammatory cytokines produced by Th17 cells) and transforming growth factor beta (TGF-β) (an immunosuppressive cytokine associated with Treg as well as Th17 differentiation3940). Results showed that upon stimulation with PDBu/Iono a great proportion of the CD3+ cells present in the spleen produced TNF and IFN-γ (Fig. 5a,b), whereas a smaller percentage produced IL-10 (Fig. 5c). Expectedly, a greater percentage of CD3+ MHCII+ cells produced TNF compared to the MHCII− T cell population (Fig. S2). Furthermore, TNF production in CD3+ T cell subsets ranged from 23.6% in Tbet− Eomes− Gata3− (representing CD4+ T cells) to 73.5% in Tbet− Eomes+ T cells (possibly representing CD8+ memory T cells) (Fig. S3a). However, those percentages varied greatly from one bat to another, again likely reflecting the differential activation status of immune cells in these wild-caught animals (Fig. S3b).


Phenotypic and functional characterization of the major lymphocyte populations in the fruit-eating bat Pteropus alecto
Bat T cell production of cytokines and cytolytic factors upon mitogenic stimulation.Bat splenocytes (a–c,g–i) or PBMCs (d–f) were stimulated for 4 h with PDBu/ionomycin or media in the presence of brefeldin A and monensin. Detection of intracellular TNF (a), IFN-γ (b), IL-10 (c) was performed at the protein level, and production of IL- 2 (d), granzyme B (e), perforin (f), IL-17a (g), IL-22 (h) and TGF-β1 (i) was detected at the mRNA level by Flow-FISH. Dot plots obtained with one bat are shown and are representative of the results obtained with 2–3 different bats.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121612&req=5

f5: Bat T cell production of cytokines and cytolytic factors upon mitogenic stimulation.Bat splenocytes (a–c,g–i) or PBMCs (d–f) were stimulated for 4 h with PDBu/ionomycin or media in the presence of brefeldin A and monensin. Detection of intracellular TNF (a), IFN-γ (b), IL-10 (c) was performed at the protein level, and production of IL- 2 (d), granzyme B (e), perforin (f), IL-17a (g), IL-22 (h) and TGF-β1 (i) was detected at the mRNA level by Flow-FISH. Dot plots obtained with one bat are shown and are representative of the results obtained with 2–3 different bats.
Mentions: To gain further insights into the functionality of P. alecto immune system, an ex-vivo mitogenic stimulation experiment was performed. Bat splenocytes or PBMCs were stimulated with PDBu and the ionophore ionomycin (activation of protein kinase C bypassing the T cell receptor), in the presence of brefeldin A and monensin that block the Golgi apparatus and allow intracellular accumulation of cytokines produced in response to the stimulation. Cross-reactive anti-human TNF and anti-human IL-10 antibodies, as well as anti-bat IFN-γ antibody were used to detect by flow cytometry the production of these cytokines. In addition, Flow-FISH was used to detect the production at the mRNA level of IL-2 (T cell growth factor), granzyme B and perforin (cytolytic molecules mainly produced by CD8+ T cells and NK cells103738), IL-17A and IL-22 (inflammatory cytokines produced by Th17 cells) and transforming growth factor beta (TGF-β) (an immunosuppressive cytokine associated with Treg as well as Th17 differentiation3940). Results showed that upon stimulation with PDBu/Iono a great proportion of the CD3+ cells present in the spleen produced TNF and IFN-γ (Fig. 5a,b), whereas a smaller percentage produced IL-10 (Fig. 5c). Expectedly, a greater percentage of CD3+ MHCII+ cells produced TNF compared to the MHCII− T cell population (Fig. S2). Furthermore, TNF production in CD3+ T cell subsets ranged from 23.6% in Tbet− Eomes− Gata3− (representing CD4+ T cells) to 73.5% in Tbet− Eomes+ T cells (possibly representing CD8+ memory T cells) (Fig. S3a). However, those percentages varied greatly from one bat to another, again likely reflecting the differential activation status of immune cells in these wild-caught animals (Fig. S3b).

View Article: PubMed Central - PubMed

ABSTRACT

The unique ability of bats to act as reservoir for viruses that are highly pathogenic to humans suggests unique properties and functional characteristics of their immune system. However, the lack of bat specific reagents, in particular antibodies, has limited our knowledge of bat’s immunity. Using cross-reactive antibodies, we report the phenotypic and functional characterization of T cell subsets, B and NK cells in the fruit-eating bat Pteropus alecto. Our findings indicate the predominance of CD8+ T cells in the spleen from wild-caught bats that may reflect either the presence of viruses in this organ or predominance of this cell subset at steady state. Instead majority of T cells in circulation, lymph nodes and bone marrow (BM) were CD4+ subsets. Interestingly, 40% of spleen T cells expressed constitutively IL-17, IL-22 and TGF-β mRNA, which may indicate a strong bias towards the Th17 and regulatory T cell subsets. Furthermore, the unexpected high number of T cells in bats BM could suggest an important role in T cell development. Finally, mitogenic stimulation induced proliferation and production of effector molecules by bats immune cells. This work contributes to a better understanding of bat’s immunity, opening up new perspectives of therapeutic interventions for humans.

No MeSH data available.