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NK and NKT Cell Depletion Alters the Outcome of Experimental Pneumococcal Pneumonia: Relationship with Regulation of Interferon-γ Production.

Christaki E, Diza E, Giamarellos-Bourboulis EJ, Papadopoulou N, Pistiki A, Droggiti DI, Georgitsi M, Machova A, Lambrelli D, Malisiovas N, Nikolaidis P, Opal SM - J Immunol Res (2015)

Bottom Line: Upon inhibition of NKT cell activation, spleen NK (CD3-/NK1.1+) cells increased compared to all other groups.Conclusions.Inhibition of NKT cell activation resulted in an increase in spleen NK (CD3-/NK1.1+) cells and a higher IFN-γ production, while altering splenocyte miRNA expression.

View Article: PubMed Central - PubMed

Affiliation: First Department of Medicine, AHEPA University Hospital, Thessaloniki, Greece ; Infectious Diseases Division, Alpert Medical School of Brown University, Providence, RI, USA.

ABSTRACT
Background. Natural killer (NK) and natural killer T (NKT) cells contribute to the innate host defense but their role in bacterial sepsis remains controversial. Methods. C57BL/6 mice were infected intratracheally with 5 × 10(5) cfu of Streptococcus pneumoniae. Animals were divided into sham group (Sham); pretreated with isotype control antibody (CON) group; pretreated with anti-asialo GM1 antibody (NKd) group; and pretreated with anti-CD1d monoclonal antibody (NKTd) group before bacterial challenge. Serum and tissue samples were analyzed for bacterial load, cytokine levels, splenocyte apoptosis rates, and cell characteristics by flow cytometry. Splenocyte miRNA expression was also analyzed and survival was assessed. Results. NK cell depletion prolonged survival. Upon inhibition of NKT cell activation, spleen NK (CD3-/NK1.1+) cells increased compared to all other groups. Inhibition of NKT cell activation led to higher bacterial loads and increased levels of serum and splenocyte IFN-γ. Splenocyte miRNA analysis showed that miR-200c and miR-29a were downregulated, while miR-125a-5p was upregulated, in anti-CD1d treated animals. These changes were moderate after NK cell depletion. Conclusions. NK cells appear to contribute to mortality in pneumococcal pneumonia. Inhibition of NKT cell activation resulted in an increase in spleen NK (CD3-/NK1.1+) cells and a higher IFN-γ production, while altering splenocyte miRNA expression.

No MeSH data available.


Related in: MedlinePlus

Cytokine production from splenocytes. Splenocytes were isolated after animal sacrifice 48 hours after bacterial challenge with Streptococcus pneumoniae. Mice were pretreated with nonspecific IgG (CON, n = 13); with anti-asialo GM1 antibody (NKd, n = 12); or with anti-CD1d antibody (NKTd, n = 10). Results are compared with sham-operated mice (Sham, n = 10). Concentrations of tumour necrosis factor-alpha (TNF-α), of interleukin (IL)-10 and of interferon-gamma (IFN-γ) were measured in supernatants after stimulation with lipopolysaccharide (LPS) of Escherichia coli O55:B5, with IL-2 and with IL-12. Serum concentrations of IFN-γ at the time of animal sacrifice are also shown. Statistically significant comparisons after corrections for multiple tests are indicated: apSham  VS  CON: 0.010; bpSham  VS  NKd: 0.033; cpSham  VS  NKTd: 0.013; dpSham  VS  NKTd: 0.049; epNKd  VS  NKTd: 0.049; fpNKd  VS  NKTd: 0.037; gpSham  VS  NKTd: 0.002; hpCON  VS  NKTd: 0.009; ipNKd  VS  NKTd: 0.039.
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fig3: Cytokine production from splenocytes. Splenocytes were isolated after animal sacrifice 48 hours after bacterial challenge with Streptococcus pneumoniae. Mice were pretreated with nonspecific IgG (CON, n = 13); with anti-asialo GM1 antibody (NKd, n = 12); or with anti-CD1d antibody (NKTd, n = 10). Results are compared with sham-operated mice (Sham, n = 10). Concentrations of tumour necrosis factor-alpha (TNF-α), of interleukin (IL)-10 and of interferon-gamma (IFN-γ) were measured in supernatants after stimulation with lipopolysaccharide (LPS) of Escherichia coli O55:B5, with IL-2 and with IL-12. Serum concentrations of IFN-γ at the time of animal sacrifice are also shown. Statistically significant comparisons after corrections for multiple tests are indicated: apSham  VS  CON: 0.010; bpSham  VS  NKd: 0.033; cpSham  VS  NKTd: 0.013; dpSham  VS  NKTd: 0.049; epNKd  VS  NKTd: 0.049; fpNKd  VS  NKTd: 0.037; gpSham  VS  NKTd: 0.002; hpCON  VS  NKTd: 0.009; ipNKd  VS  NKTd: 0.039.

Mentions: Prolonged survival after depletion of NK cells was related to reduced outgrowth of S. pneumoniae in the lung; the opposite was observed in animals where CD1d activation of NKT cells was blocked (Figure 2). In order to investigate a mechanistic explanation behind the survival benefit in the animals depleted of NK cells and disadvantage in those deprived of CD1d-dependent NKT cell activation, we hypothesized that an imbalance of NK/NKT cells may affect the local and systemic dissemination of the inoculated pathogen. A change in NK and NKT cell populations could potentially influence the apoptosis of tissue macrophages or it could affect the type of cytokine profile that determines the host innate immune response to S. pneumoniae. Depletion of NK cells and blockade of CD1d-dependent NKT cell activation did not change the rate of spleen lymphocyte and macrophage apoptosis (data not shown). Also, there were no significant differences between the groups in regard to TNF-α and IL-10 production by splenocytes after stimulation with LPS, IL-2, and IL-12, although there was a trend for decreased IL-10 production after stimulation with IL-12 in the anti-asialo GM1 treated group compared with controls. However, when splenocytes were stimulated ex vivo, production of IFN-γ differed between the animal groups. More precisely, in the event of inhibition of NKT cell activation, that is, of predominance of NK cells, an exaggerated production of IFN-γ was found (Figure 3). This was also reflected in the concentrations of circulating IFN-γ (Figure 3).


NK and NKT Cell Depletion Alters the Outcome of Experimental Pneumococcal Pneumonia: Relationship with Regulation of Interferon-γ Production.

Christaki E, Diza E, Giamarellos-Bourboulis EJ, Papadopoulou N, Pistiki A, Droggiti DI, Georgitsi M, Machova A, Lambrelli D, Malisiovas N, Nikolaidis P, Opal SM - J Immunol Res (2015)

Cytokine production from splenocytes. Splenocytes were isolated after animal sacrifice 48 hours after bacterial challenge with Streptococcus pneumoniae. Mice were pretreated with nonspecific IgG (CON, n = 13); with anti-asialo GM1 antibody (NKd, n = 12); or with anti-CD1d antibody (NKTd, n = 10). Results are compared with sham-operated mice (Sham, n = 10). Concentrations of tumour necrosis factor-alpha (TNF-α), of interleukin (IL)-10 and of interferon-gamma (IFN-γ) were measured in supernatants after stimulation with lipopolysaccharide (LPS) of Escherichia coli O55:B5, with IL-2 and with IL-12. Serum concentrations of IFN-γ at the time of animal sacrifice are also shown. Statistically significant comparisons after corrections for multiple tests are indicated: apSham  VS  CON: 0.010; bpSham  VS  NKd: 0.033; cpSham  VS  NKTd: 0.013; dpSham  VS  NKTd: 0.049; epNKd  VS  NKTd: 0.049; fpNKd  VS  NKTd: 0.037; gpSham  VS  NKTd: 0.002; hpCON  VS  NKTd: 0.009; ipNKd  VS  NKTd: 0.039.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: Cytokine production from splenocytes. Splenocytes were isolated after animal sacrifice 48 hours after bacterial challenge with Streptococcus pneumoniae. Mice were pretreated with nonspecific IgG (CON, n = 13); with anti-asialo GM1 antibody (NKd, n = 12); or with anti-CD1d antibody (NKTd, n = 10). Results are compared with sham-operated mice (Sham, n = 10). Concentrations of tumour necrosis factor-alpha (TNF-α), of interleukin (IL)-10 and of interferon-gamma (IFN-γ) were measured in supernatants after stimulation with lipopolysaccharide (LPS) of Escherichia coli O55:B5, with IL-2 and with IL-12. Serum concentrations of IFN-γ at the time of animal sacrifice are also shown. Statistically significant comparisons after corrections for multiple tests are indicated: apSham  VS  CON: 0.010; bpSham  VS  NKd: 0.033; cpSham  VS  NKTd: 0.013; dpSham  VS  NKTd: 0.049; epNKd  VS  NKTd: 0.049; fpNKd  VS  NKTd: 0.037; gpSham  VS  NKTd: 0.002; hpCON  VS  NKTd: 0.009; ipNKd  VS  NKTd: 0.039.
Mentions: Prolonged survival after depletion of NK cells was related to reduced outgrowth of S. pneumoniae in the lung; the opposite was observed in animals where CD1d activation of NKT cells was blocked (Figure 2). In order to investigate a mechanistic explanation behind the survival benefit in the animals depleted of NK cells and disadvantage in those deprived of CD1d-dependent NKT cell activation, we hypothesized that an imbalance of NK/NKT cells may affect the local and systemic dissemination of the inoculated pathogen. A change in NK and NKT cell populations could potentially influence the apoptosis of tissue macrophages or it could affect the type of cytokine profile that determines the host innate immune response to S. pneumoniae. Depletion of NK cells and blockade of CD1d-dependent NKT cell activation did not change the rate of spleen lymphocyte and macrophage apoptosis (data not shown). Also, there were no significant differences between the groups in regard to TNF-α and IL-10 production by splenocytes after stimulation with LPS, IL-2, and IL-12, although there was a trend for decreased IL-10 production after stimulation with IL-12 in the anti-asialo GM1 treated group compared with controls. However, when splenocytes were stimulated ex vivo, production of IFN-γ differed between the animal groups. More precisely, in the event of inhibition of NKT cell activation, that is, of predominance of NK cells, an exaggerated production of IFN-γ was found (Figure 3). This was also reflected in the concentrations of circulating IFN-γ (Figure 3).

Bottom Line: Upon inhibition of NKT cell activation, spleen NK (CD3-/NK1.1+) cells increased compared to all other groups.Conclusions.Inhibition of NKT cell activation resulted in an increase in spleen NK (CD3-/NK1.1+) cells and a higher IFN-γ production, while altering splenocyte miRNA expression.

View Article: PubMed Central - PubMed

Affiliation: First Department of Medicine, AHEPA University Hospital, Thessaloniki, Greece ; Infectious Diseases Division, Alpert Medical School of Brown University, Providence, RI, USA.

ABSTRACT
Background. Natural killer (NK) and natural killer T (NKT) cells contribute to the innate host defense but their role in bacterial sepsis remains controversial. Methods. C57BL/6 mice were infected intratracheally with 5 × 10(5) cfu of Streptococcus pneumoniae. Animals were divided into sham group (Sham); pretreated with isotype control antibody (CON) group; pretreated with anti-asialo GM1 antibody (NKd) group; and pretreated with anti-CD1d monoclonal antibody (NKTd) group before bacterial challenge. Serum and tissue samples were analyzed for bacterial load, cytokine levels, splenocyte apoptosis rates, and cell characteristics by flow cytometry. Splenocyte miRNA expression was also analyzed and survival was assessed. Results. NK cell depletion prolonged survival. Upon inhibition of NKT cell activation, spleen NK (CD3-/NK1.1+) cells increased compared to all other groups. Inhibition of NKT cell activation led to higher bacterial loads and increased levels of serum and splenocyte IFN-γ. Splenocyte miRNA analysis showed that miR-200c and miR-29a were downregulated, while miR-125a-5p was upregulated, in anti-CD1d treated animals. These changes were moderate after NK cell depletion. Conclusions. NK cells appear to contribute to mortality in pneumococcal pneumonia. Inhibition of NKT cell activation resulted in an increase in spleen NK (CD3-/NK1.1+) cells and a higher IFN-γ production, while altering splenocyte miRNA expression.

No MeSH data available.


Related in: MedlinePlus