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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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Depletion of NK cells in NK-/-EM/IOE mice decreased production of iNOS and NO.Spleen cells 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. (A) Dot plots shows negative control (cells stained with isotype control Ab) and positive control (splenocytes from IOE-infected mice stimulated in vitro with LPS). Cells were gated on leukocytes based on forward and side scatter. (B and C) Data show lower percentages and absolute number, respectively, of iNOS-producing leukocytes compared with NK+/+EM/IOE mice. (D) The level of NO produced by E. muris-stimulated cells was measured as described in Materials and Methods. Data show a significantly lower production of NO by splenocytes from NK-/-EM/IOE mice compared with NK+/+EM/IOE mice. The levels of NO in NK-/-EM/IOE-mice were similar to those detected in naïve mice infected with IOE. * and ** indicate P < 0.05 and P < 0.01, respectively. Data are representative of two independent experiments with four mice per group.
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pone.0153223.g010: Depletion of NK cells in NK-/-EM/IOE mice decreased production of iNOS and NO.Spleen cells 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. (A) Dot plots shows negative control (cells stained with isotype control Ab) and positive control (splenocytes from IOE-infected mice stimulated in vitro with LPS). Cells were gated on leukocytes based on forward and side scatter. (B and C) Data show lower percentages and absolute number, respectively, of iNOS-producing leukocytes compared with NK+/+EM/IOE mice. (D) The level of NO produced by E. muris-stimulated cells was measured as described in Materials and Methods. Data show a significantly lower production of NO by splenocytes from NK-/-EM/IOE mice compared with NK+/+EM/IOE mice. The levels of NO in NK-/-EM/IOE-mice were similar to those detected in naïve mice infected with IOE. * and ** indicate P < 0.05 and P < 0.01, respectively. Data are representative of two independent experiments with four mice per group.

Mentions: IFN-γ is known to mediate activation of the bactericidal functions of macrophages, such as the induction of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO). The latter is a key antimicrobial effector molecule. Thus, we examined whether the difference in IFN-γ response between NK depleted and NK sufficient mice influenced the production of iNOS and NO. We measured the number of CD45+ leukocytes producing iNOS and the quantity of nitrites in the culture supernatants by flow cytometry and Griess reaction, respectively. Negligible iNOS production was detected in isotype control mAb- stained splenocytes from IOE-infected mice (negative control) Fig 10A, while a significantly high percentage of iNOS-producing cells were detected when IOE-infected splenocytes were further stimulated in vitro with lipopolysaccharide (LPS) (positive control) Fig 10A. Our data showed that the spleens of NK+/+EM/IOE-infected mice contain a higher percentage Fig 10B and absolute number Fig 10C of iNOS-producing leukocytes when compared to IOE-infected mice and NK-/-EM/IOE. Consistent with single cell analysis, there was a higher production of nitrite in spleen bulk culture in NK+/+EM/IOE-infected mice compared to other mice groups Fig 10D. Together, these data suggest that NK cells promote bacterial elimination and the protective memory response against Ehrlichia by enhancing the induction and/or expansion of effector memory CD4+ Th1 cells, IFN-γ production, and the activation of the bactericidal function of infected phagocytic cells.


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

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

Depletion of NK cells in NK-/-EM/IOE mice decreased production of iNOS and NO.Spleen cells 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. (A) Dot plots shows negative control (cells stained with isotype control Ab) and positive control (splenocytes from IOE-infected mice stimulated in vitro with LPS). Cells were gated on leukocytes based on forward and side scatter. (B and C) Data show lower percentages and absolute number, respectively, of iNOS-producing leukocytes compared with NK+/+EM/IOE mice. (D) The level of NO produced by E. muris-stimulated cells was measured as described in Materials and Methods. Data show a significantly lower production of NO by splenocytes from NK-/-EM/IOE mice compared with NK+/+EM/IOE mice. The levels of NO in NK-/-EM/IOE-mice were similar to those detected in naïve mice infected with IOE. * and ** indicate P < 0.05 and P < 0.01, respectively. Data are representative of two independent experiments with four mice per group.
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pone.0153223.g010: Depletion of NK cells in NK-/-EM/IOE mice decreased production of iNOS and NO.Spleen cells 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. (A) Dot plots shows negative control (cells stained with isotype control Ab) and positive control (splenocytes from IOE-infected mice stimulated in vitro with LPS). Cells were gated on leukocytes based on forward and side scatter. (B and C) Data show lower percentages and absolute number, respectively, of iNOS-producing leukocytes compared with NK+/+EM/IOE mice. (D) The level of NO produced by E. muris-stimulated cells was measured as described in Materials and Methods. Data show a significantly lower production of NO by splenocytes from NK-/-EM/IOE mice compared with NK+/+EM/IOE mice. The levels of NO in NK-/-EM/IOE-mice were similar to those detected in naïve mice infected with IOE. * and ** indicate P < 0.05 and P < 0.01, respectively. Data are representative of two independent experiments with four mice per group.
Mentions: IFN-γ is known to mediate activation of the bactericidal functions of macrophages, such as the induction of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO). The latter is a key antimicrobial effector molecule. Thus, we examined whether the difference in IFN-γ response between NK depleted and NK sufficient mice influenced the production of iNOS and NO. We measured the number of CD45+ leukocytes producing iNOS and the quantity of nitrites in the culture supernatants by flow cytometry and Griess reaction, respectively. Negligible iNOS production was detected in isotype control mAb- stained splenocytes from IOE-infected mice (negative control) Fig 10A, while a significantly high percentage of iNOS-producing cells were detected when IOE-infected splenocytes were further stimulated in vitro with lipopolysaccharide (LPS) (positive control) Fig 10A. Our data showed that the spleens of NK+/+EM/IOE-infected mice contain a higher percentage Fig 10B and absolute number Fig 10C of iNOS-producing leukocytes when compared to IOE-infected mice and NK-/-EM/IOE. Consistent with single cell analysis, there was a higher production of nitrite in spleen bulk culture in NK+/+EM/IOE-infected mice compared to other mice groups Fig 10D. Together, these data suggest that NK cells promote bacterial elimination and the protective memory response against Ehrlichia by enhancing the induction and/or expansion of effector memory CD4+ Th1 cells, IFN-γ production, and the activation of the bactericidal function of infected phagocytic cells.

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