The transcription factor nuclear factor of activated T cells c3 modulates the function of macrophages in sepsis

Abstract

The role of the transcription factor nuclear factor of activated T cells (NFAT) was initially identified in T and B cell gene expression, but its role in regulating gene expression in macrophages during sepsis is not known. Our data show that NFATc3 regulates expression of inducible nitric oxide synthase (iNOS) in macrophages stimulated with lipopolysaccharide. Selective inhibition of NFAT by cyclosporine A and a competitive peptide inhibitor 11R-VIVIT inhibited endotoxin-induced expression of iNOS and nitric oxide (NO) release. Macrophages from NFATc3 knockout (KO) mice show reduced iNOS expression and NO release and attenuated bactericidal activity. Gel shift and chromatin immunoprecipitation assays show that endotoxin challenge increases NFATc3 binding to the iNOS promoter, resulting in transcriptional activation of iNOS. The binding of NFATc3 to the iNOS promoter is abolished by NFAT inhibitors. NFATc3 KO mice subjected to sepsis show that NFATc3 is necessary for bacterial clearance in mouse lungs during sepsis. Our study demonstrates for the first time that NFATc3 is necessary for macrophage iNOS expression during sepsis, which is essential for containment of bacterial infections.

Fig. 1

Expression of iNOS in macrophages is regulated by NFAT. BMDM-Luc were treated with LPS (100 ng/ml), and expression of iNOS was determined at various time points. a, b iNOS mRNA levels were determined by RT-qPCR (a), and protein level was determined by Western blot (b). Expression peaked at approximately 8-16 h following treatment with LPS. c, e Macrophages pretreated with specific inhibitors of NFAT (CsA and 11R-VIVIT, respectively) and challenged with LPS showed significant inhibition of expression of iNOS (c, e, representative Western blots). d, f The concentration of nitrite in the cell culture media, which is an indicator of iNOS expression, was also measured. The concentration of nitrite in the presence of NFAT inhibitors CsA (d) and 11R-VIVIT (f) was analyzed. Treatment with specific NFAT inhibitors resulted in significant reduction of nitrite in cell culture media, indicating that the expression of iNOS was reduced. 11R-VEET was used as control. The error bars represent the SEM from 3 independent experiments. * p ≤ 0.05, ** p ≤ 0.005. UT = Untreated.

Fig. 2

LPS causes NFATc3 nuclear translocation specifically, which regulates the iNOS expression. a BMDM were treated with 100 ng/ml LPS for the indicated time periods, and cells were harvested. Cytosolic and nuclear protein fractions were resolved and analyzed by immunoblotting with NFATc1, NFATc2, NFATc3 and NFATc4 antibodies. b WT (NFATc3^+/+) and NFATc3^−/− BMDM were stimulated with 100 ng/ml LPS and analyzed for iNOS expression as described in figure 1b. c WT (NFATc3^+/+) and NFATc3^−/− BMDM were stimulated with 100 ng/ml LPS and analyzed for NO release as described in figure 1d and f. * p ≤ 0.05, ** p ≤ 0.005.

Fig. 3

DNA binding activity of NFATc3 is directly involved in transcriptional regulation of iNOS. a Schematic representation of the iNOS promoter (approx. 2-kb genomic region) representing cognate NFATc3 binding sites. b BMDM were treated with LPS and/or CsA for 30 min; nuclear extracts were prepared as described in Materials and Methods, and an equal amount of extract was incubated with biotin-labeled double-stranded DNA containing a consensus binding site for NFAT. The NFAT protein-DNA complex was resolved on a 5% native acrylamide gel, transferred onto a Nylon membrane and developed using a Pierce chemiluminescent detection kit. c Equal amounts of nuclear extract protein were incubated with biotin-labeled double-stranded DNA containing a consensus binding site for NFAT, competing against the unlabeled (cold) probe in increasing molar concentrations and processed as described in (b). d The specificity of DNA binding was determined using NFATc3 antibody, included in the oligo-protein reaction mixture. The black arrow represents the super shift band, and white arrows represent binding activity of the NFAT probe. e, f Recruitment of NFATc3 to iNOS promoter was assessed by ChIP. The eluted DNA was subjected to real-time PCR, and the abundance of NFATc3 (e) and RNA Polymerase II (RNA-Pol-II; f) at the iNOS promoter was calculated using the ratio of amplification efficiency (crossing point value) of the sample over nonimmune IgG. UT = Untreated.

Fig. 4

Deficiency of NFATc3 results in attenuated bactericidal activity. a WT macrophages were treated with 11R-VIVIT (specific NFAT inhibitor) and incubated with E. coli, and bactericidal activity was analyzed by plating macrophage lysate on bacterial culture plates. The NFAT inhibitor-treated macrophages had attenuated bactericidal activity compared to the group treated with control peptide (11R-VEET). b Macrophages cultured from WT and NFATc3 KO mice were treated with E. coli, and bactericidal activity was analyzed by plating macrophage lysate on bacterial culture plates. The NFATc3-deficient macrophages had significantly attenuated bactericidal activity compared to WT macrophages. Error bars represent the standard deviation. * p < 0.01. n = 3.

Fig. 5

Inhibition of NFATc3 results in bacterial infection in lungs during sepsis. The WT and NFATc3 KO mice were subjected to CLP. After 24 h the mice were euthanized, and bacterial infection in the lungs was assessed by plating lung homogenate on sheep blood agar plates. The NFATc3 KO mice subjected to sepsis by CLP had severe bacterial infection compared to WT mice. Error bars represent the standard deviation. * p < 0.01. n = 3.

Fig. 6

Transcription factor NFATc3 regulates macrophage bactericidal activity. During the onset of sepsis by bacterial infection, the transcription factor NFATc3 in macrophages is activated by the change in the calcium levels, which results in its translocation to the nucleus, resulting in the transcription of iNOS. This results in robust generation of NO and other reactive oxygen/nitrogen intermediates, resulting in efficient bactericidal activity and thus contributing to the resolution of harmful bacterial infection and inflammation.