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NK Cell-Mediated Regulation of Protective Memory Responses against Intracellular Ehrlichial Pathogens.

Habib S, El Andaloussi A, Hisham A, Ismail N - PLoS ONE (2016)

Bottom Line: Ehrlichiae are gram-negative obligate intracellular bacteria that cause potentially fatal human monocytic ehrlichiosis.However, the contribution of NK cells to the memory response against Ehrlichia remains elusive.Together, these data suggest that E. muris-induced memory-like NK cells, which contribute to the protective, recall response against Ehrlichia.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Medical College of Georgia, Augusta University, Augusta, Georgia, United States of America.

ABSTRACT
Ehrlichiae are gram-negative obligate intracellular bacteria that cause potentially fatal human monocytic ehrlichiosis. We previously showed that natural killer (NK) cells play a critical role in host defense against Ehrlichia during primary infection. However, the contribution of NK cells to the memory response against Ehrlichia remains elusive. Primary infection of C57BL/6 mice with Ehrlichia muris provides long-term protection against a second challenge with the highly virulent Ixodes ovatus Ehrlichia (IOE), which ordinarily causes fatal disease in naïve mice. Here, we show that the depletion of NK cells in E. muris-primed mice abrogates the protective memory response against IOE. Approximately, 80% of NK cell-depleted E. muris-primed mice succumbed to lethal IOE infection on days 8-10 after IOE infection, similar to naïve mice infected with the same dose of IOE. The lack of a recall response in NK cell-depleted mice correlated with an increased bacterial burden, extensive liver injury, decreased frequency of Ehrlichia-specific IFN-γ-producing memory CD4+ and CD8+ T-cells, and a low titer of Ehrlichia-specific antibodies. Intraperitoneal infection of mice with E. muris resulted in the production of IL-15, IL-12, and IFN-γ as well as an expansion of activated NKG2D+ NK cells. The adoptive transfer of purified E. muris-primed hepatic and splenic NK cells into Rag2-/-Il2rg-/- recipient mice provided protective immunity against challenge with E. muris. Together, these data suggest that E. muris-induced memory-like NK cells, which contribute to the protective, recall response against Ehrlichia.

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Decreased percentage of IFN-γ producing CD3+ T cells in NK-/-EM/IOE-mice.Splenocytes were harvested from the indicated mice groups on day 7 after IOE infection, and were stimulated in vitro with E. muris Ags or left unstimulated. Lymphocytes were gated based on the forward and side scatter, and then cells were gated on CD3. CD3+ T cells were further analyzed for intracellular IFN-γ staining as shown in the gating strategy (A). Controls shown in dot plots includes naïve splenocytes stimulated in vitro with E. muris Ags and IFN-γ production by unstimulated splenocytes from IOE-infected mice. (B) Dot plots shows the percentage of gated CD3+ T cells and the percentage of IFN-γ+ CD3+ T cell subset in indicated mice groups following in vitro stimulation with E. muris Ags. (C) Data show the differences in the absolute number of IFN-γ+ CD3+ T cells in the spleen of indicated mice groups. Data shown are from three mice/groups with similar results in three independent experiments (n = 9 mice/group).
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pone.0153223.g007: Decreased percentage of IFN-γ producing CD3+ T cells in NK-/-EM/IOE-mice.Splenocytes were harvested from the indicated mice groups on day 7 after IOE infection, and were stimulated in vitro with E. muris Ags or left unstimulated. Lymphocytes were gated based on the forward and side scatter, and then cells were gated on CD3. CD3+ T cells were further analyzed for intracellular IFN-γ staining as shown in the gating strategy (A). Controls shown in dot plots includes naïve splenocytes stimulated in vitro with E. muris Ags and IFN-γ production by unstimulated splenocytes from IOE-infected mice. (B) Dot plots shows the percentage of gated CD3+ T cells and the percentage of IFN-γ+ CD3+ T cell subset in indicated mice groups following in vitro stimulation with E. muris Ags. (C) Data show the differences in the absolute number of IFN-γ+ CD3+ T cells in the spleen of indicated mice groups. Data shown are from three mice/groups with similar results in three independent experiments (n = 9 mice/group).

Mentions: IFN-γ is critical for effective bacterial elimination in ehrlichiosis. T cells, particularly CD4+ Th1 cells and NKT cells are major cellular subsets that produce IFN-γ and thus mediate protective immunity against Ehrlichia. To determine the role of NK cells in host defense against Ehrlichia during recall response, we examined the number of antigen-specific, IFN-γ-producing T cells and NKT cells in all mice groups on day 7 after the second IOE infection. We stimulated splenocytes from naïve and infected mice with E. muris Ags, and the frequency of E. muris-specific, IFN-γ-producing cells was determined by flow cytometry. Impaired protective immunity in NK-/-EM/IOE mice was associated with decreases in the percentage Fig 7A and 7B and absolute number Fig 7C of E. muris-specific IFN-γ-producing CD3+ T cells when compared with those found in NK+/+EM/IOE control mice. Similarly, the percentage Fig 8A and 8B and absolute number Fig 8C of IFN-γ-producing NKT cells was decreased in NK-/-EM/IOE mice compared to NK+/+EM/IOE mice. Polyclonal stimulation of splenocytes from all mice (naïve and infected) groups with PMA/ionomycin resulted in a similar increase in the number of IFN-γ-producing T cells (data not shown), suggesting that an impaired memory CD4+ Th1 response in NK-/- EM/IOE mice encompasses antigen-specific T cells. We did not find a significant difference in the frequency of antigen-specific, IL-4-producing cells or IL-10-producing T cells between NK+/+EM/IOE and NK-/-EM/IOE-infected mice (data not shown), suggesting that impaired bacterial elimination is not due to altered IL-4: IFN-γ or IL-10:IFN-γ responses, as suggested by other studies [18,21,26,27].


NK Cell-Mediated Regulation of Protective Memory Responses against Intracellular Ehrlichial Pathogens.

Habib S, El Andaloussi A, Hisham A, Ismail N - PLoS ONE (2016)

Decreased percentage of IFN-γ producing CD3+ T cells in NK-/-EM/IOE-mice.Splenocytes were harvested from the indicated mice groups on day 7 after IOE infection, and were stimulated in vitro with E. muris Ags or left unstimulated. Lymphocytes were gated based on the forward and side scatter, and then cells were gated on CD3. CD3+ T cells were further analyzed for intracellular IFN-γ staining as shown in the gating strategy (A). Controls shown in dot plots includes naïve splenocytes stimulated in vitro with E. muris Ags and IFN-γ production by unstimulated splenocytes from IOE-infected mice. (B) Dot plots shows the percentage of gated CD3+ T cells and the percentage of IFN-γ+ CD3+ T cell subset in indicated mice groups following in vitro stimulation with E. muris Ags. (C) Data show the differences in the absolute number of IFN-γ+ CD3+ T cells in the spleen of indicated mice groups. Data shown are from three mice/groups with similar results in three independent experiments (n = 9 mice/group).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4836677&req=5

pone.0153223.g007: Decreased percentage of IFN-γ producing CD3+ T cells in NK-/-EM/IOE-mice.Splenocytes were harvested from the indicated mice groups on day 7 after IOE infection, and were stimulated in vitro with E. muris Ags or left unstimulated. Lymphocytes were gated based on the forward and side scatter, and then cells were gated on CD3. CD3+ T cells were further analyzed for intracellular IFN-γ staining as shown in the gating strategy (A). Controls shown in dot plots includes naïve splenocytes stimulated in vitro with E. muris Ags and IFN-γ production by unstimulated splenocytes from IOE-infected mice. (B) Dot plots shows the percentage of gated CD3+ T cells and the percentage of IFN-γ+ CD3+ T cell subset in indicated mice groups following in vitro stimulation with E. muris Ags. (C) Data show the differences in the absolute number of IFN-γ+ CD3+ T cells in the spleen of indicated mice groups. Data shown are from three mice/groups with similar results in three independent experiments (n = 9 mice/group).
Mentions: IFN-γ is critical for effective bacterial elimination in ehrlichiosis. T cells, particularly CD4+ Th1 cells and NKT cells are major cellular subsets that produce IFN-γ and thus mediate protective immunity against Ehrlichia. To determine the role of NK cells in host defense against Ehrlichia during recall response, we examined the number of antigen-specific, IFN-γ-producing T cells and NKT cells in all mice groups on day 7 after the second IOE infection. We stimulated splenocytes from naïve and infected mice with E. muris Ags, and the frequency of E. muris-specific, IFN-γ-producing cells was determined by flow cytometry. Impaired protective immunity in NK-/-EM/IOE mice was associated with decreases in the percentage Fig 7A and 7B and absolute number Fig 7C of E. muris-specific IFN-γ-producing CD3+ T cells when compared with those found in NK+/+EM/IOE control mice. Similarly, the percentage Fig 8A and 8B and absolute number Fig 8C of IFN-γ-producing NKT cells was decreased in NK-/-EM/IOE mice compared to NK+/+EM/IOE mice. Polyclonal stimulation of splenocytes from all mice (naïve and infected) groups with PMA/ionomycin resulted in a similar increase in the number of IFN-γ-producing T cells (data not shown), suggesting that an impaired memory CD4+ Th1 response in NK-/- EM/IOE mice encompasses antigen-specific T cells. We did not find a significant difference in the frequency of antigen-specific, IL-4-producing cells or IL-10-producing T cells between NK+/+EM/IOE and NK-/-EM/IOE-infected mice (data not shown), suggesting that impaired bacterial elimination is not due to altered IL-4: IFN-γ or IL-10:IFN-γ responses, as suggested by other studies [18,21,26,27].

Bottom Line: Ehrlichiae are gram-negative obligate intracellular bacteria that cause potentially fatal human monocytic ehrlichiosis.However, the contribution of NK cells to the memory response against Ehrlichia remains elusive.Together, these data suggest that E. muris-induced memory-like NK cells, which contribute to the protective, recall response against Ehrlichia.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Medical College of Georgia, Augusta University, Augusta, Georgia, United States of America.

ABSTRACT
Ehrlichiae are gram-negative obligate intracellular bacteria that cause potentially fatal human monocytic ehrlichiosis. We previously showed that natural killer (NK) cells play a critical role in host defense against Ehrlichia during primary infection. However, the contribution of NK cells to the memory response against Ehrlichia remains elusive. Primary infection of C57BL/6 mice with Ehrlichia muris provides long-term protection against a second challenge with the highly virulent Ixodes ovatus Ehrlichia (IOE), which ordinarily causes fatal disease in naïve mice. Here, we show that the depletion of NK cells in E. muris-primed mice abrogates the protective memory response against IOE. Approximately, 80% of NK cell-depleted E. muris-primed mice succumbed to lethal IOE infection on days 8-10 after IOE infection, similar to naïve mice infected with the same dose of IOE. The lack of a recall response in NK cell-depleted mice correlated with an increased bacterial burden, extensive liver injury, decreased frequency of Ehrlichia-specific IFN-γ-producing memory CD4+ and CD8+ T-cells, and a low titer of Ehrlichia-specific antibodies. Intraperitoneal infection of mice with E. muris resulted in the production of IL-15, IL-12, and IFN-γ as well as an expansion of activated NKG2D+ NK cells. The adoptive transfer of purified E. muris-primed hepatic and splenic NK cells into Rag2-/-Il2rg-/- recipient mice provided protective immunity against challenge with E. muris. Together, these data suggest that E. muris-induced memory-like NK cells, which contribute to the protective, recall response against Ehrlichia.

Show MeSH
Related in: MedlinePlus