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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

Expression of miRNA and of mRNA of IFN-γ in splenocytes. Splenocyte total RNA was extracted. (a) Τhe expression levels of miR-155, miR-200ca, miR-29a, and miR-125a-5p were quantified by TaqMan miRNA assays. Data was normalized to the endogenous control sno202 and fold changes of miRNA expression relative to the CON group were calculated by the ΔΔCT method. (b) Expression of IFN-γ mRNA in splenocytes. Splenocyte total RNA was extracted and the expression level of IFN-γ was quantified by TaqMan assays. Data was normalized to the endogenous control GAPDH and fold changes of mRNA expression relative to the CON group were calculated by the ΔΔCT method. Mice were pretreated with nonspecific IgG (CON, n = 1); with anti-asialo GM1 antibody (NKd, n = 2); or with anti-CD1d antibody (NKTd, n = 2). Results are compared with sham-operated mice (Sham, n = 1).
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fig4: Expression of miRNA and of mRNA of IFN-γ in splenocytes. Splenocyte total RNA was extracted. (a) Τhe expression levels of miR-155, miR-200ca, miR-29a, and miR-125a-5p were quantified by TaqMan miRNA assays. Data was normalized to the endogenous control sno202 and fold changes of miRNA expression relative to the CON group were calculated by the ΔΔCT method. (b) Expression of IFN-γ mRNA in splenocytes. Splenocyte total RNA was extracted and the expression level of IFN-γ was quantified by TaqMan assays. Data was normalized to the endogenous control GAPDH and fold changes of mRNA expression relative to the CON group were calculated by the ΔΔCT method. Mice were pretreated with nonspecific IgG (CON, n = 1); with anti-asialo GM1 antibody (NKd, n = 2); or with anti-CD1d antibody (NKTd, n = 2). Results are compared with sham-operated mice (Sham, n = 1).

Mentions: To further investigate the mechanism that may regulate the increased IFN-γ production observed upon blockade of CD1d activation of NKT cells during sepsis, we compared the expression of IFN-γ mRNA and of specific immune-related miRNAs in splenocytes between samples from the four different groups. Interestingly, miR-155, miR-200c, and miR-29a were found to be downregulated in the splenocytes of the NKTd group, by 60% to 80% compared to controls (Figure 4(a)). In contrast, miR-125a-5p was upregulated in NKTd splenocytes 8-fold compared to all other groups. Following quantitation of IFN-γ mRNA in the same samples, we found a 5-fold increase in the NKTd animals (Figure 4(b)), which correlates negatively to miR-155, miR-200c, and miR-29a downregulation and positively to miR-125a-5p induction. These data provide an indication that inhibition of CD1d-dependent NKT cell activation may be associated with specific miRNA alterations during sepsis, which may be implicated in the transcriptional regulation of IFN-γ production.


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)

Expression of miRNA and of mRNA of IFN-γ in splenocytes. Splenocyte total RNA was extracted. (a) Τhe expression levels of miR-155, miR-200ca, miR-29a, and miR-125a-5p were quantified by TaqMan miRNA assays. Data was normalized to the endogenous control sno202 and fold changes of miRNA expression relative to the CON group were calculated by the ΔΔCT method. (b) Expression of IFN-γ mRNA in splenocytes. Splenocyte total RNA was extracted and the expression level of IFN-γ was quantified by TaqMan assays. Data was normalized to the endogenous control GAPDH and fold changes of mRNA expression relative to the CON group were calculated by the ΔΔCT method. Mice were pretreated with nonspecific IgG (CON, n = 1); with anti-asialo GM1 antibody (NKd, n = 2); or with anti-CD1d antibody (NKTd, n = 2). Results are compared with sham-operated mice (Sham, n = 1).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: Expression of miRNA and of mRNA of IFN-γ in splenocytes. Splenocyte total RNA was extracted. (a) Τhe expression levels of miR-155, miR-200ca, miR-29a, and miR-125a-5p were quantified by TaqMan miRNA assays. Data was normalized to the endogenous control sno202 and fold changes of miRNA expression relative to the CON group were calculated by the ΔΔCT method. (b) Expression of IFN-γ mRNA in splenocytes. Splenocyte total RNA was extracted and the expression level of IFN-γ was quantified by TaqMan assays. Data was normalized to the endogenous control GAPDH and fold changes of mRNA expression relative to the CON group were calculated by the ΔΔCT method. Mice were pretreated with nonspecific IgG (CON, n = 1); with anti-asialo GM1 antibody (NKd, n = 2); or with anti-CD1d antibody (NKTd, n = 2). Results are compared with sham-operated mice (Sham, n = 1).
Mentions: To further investigate the mechanism that may regulate the increased IFN-γ production observed upon blockade of CD1d activation of NKT cells during sepsis, we compared the expression of IFN-γ mRNA and of specific immune-related miRNAs in splenocytes between samples from the four different groups. Interestingly, miR-155, miR-200c, and miR-29a were found to be downregulated in the splenocytes of the NKTd group, by 60% to 80% compared to controls (Figure 4(a)). In contrast, miR-125a-5p was upregulated in NKTd splenocytes 8-fold compared to all other groups. Following quantitation of IFN-γ mRNA in the same samples, we found a 5-fold increase in the NKTd animals (Figure 4(b)), which correlates negatively to miR-155, miR-200c, and miR-29a downregulation and positively to miR-125a-5p induction. These data provide an indication that inhibition of CD1d-dependent NKT cell activation may be associated with specific miRNA alterations during sepsis, which may be implicated in the transcriptional regulation of IFN-γ production.

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