NF-κBp50 and HDAC1 Interaction Is Implicated in the Host Tolerance to Infection Mediated by the Bacterial Quorum Sensing Signal 2-Aminoacetophenone

Abstract

Some bacterial quorum sensing (QS) small molecules are important mediators of inter-kingdom signaling and impact host immunity. The QS regulated small volatile molecule 2-aminoacetophenone (2-AA), which has been proposed as a biomarker of Pseudomonas aeruginosa colonization in chronically infected human tissues, is critically involved in "host tolerance training" that involves a distinct molecular mechanism of host chromatin regulation through histone deacetylase (HDAC)1. 2-AA's epigenetic reprogramming action enables host tolerance to high bacterial burden and permits long-term presence of P. aeruginosa without compromising host survival. Here, to further elucidate the molecular mechanisms of 2-AA-mediated host tolerance/resilience we investigated the connection between histone acetylation status and nuclear factor (NF)-κB signaling components that together coordinate 2-AA-mediated control of transcriptional activity. We found increased NF-κBp65 acetylation levels in 2-AA stimulated cells that are preceded by association of CBP/p300 and increased histone acetyltransferase activity. In contrast, in 2-AA-tolerized cells the protein-protein interaction between p65 and CBP/p300 is disrupted and conversely, the interaction between p50 and co-repressor HDAC1 is enhanced, leading to repression of the pro-inflammatory response. These results highlight how a bacterial QS signaling molecule can establish a link between intracellular signaling and epigenetic reprogramming of pro-inflammatory mediators that may contribute to host tolerance training. These new insights might contribute to the development of novel therapeutic interventions against bacterial infections.

Keywords: 2-aminoacetophenone; CBP/p300; HDAC1; NF-κB; inflammatory cytokines; quorum sensing.

FIGURE 1

2-AA-mediated immunomodulation. Our studies have shown that 2-AA tolerization dampens the activation of NF-κB pathway (1) (Bandyopadhaya et al., 2012). The DNA binding activity of NF-κB p65 (2) (Bandyopadhaya et al., 2012) is impaired due to reduced acetylation level of p65 in 2-AA-tolerized cells (3 this study). 2-AA tolerization reduces histone acetylation (H3) via downregulation of CBP/p300 HAT activity and expression (4 this study) and the protein–protein interaction between p65 and CBP/p300 (5 this study). The upregulation of HDAC1 initiates HDAC1–p50 interaction and p50 homodimer–HDAC1 complexes are recruited at targeted sites (6 this study). Consequently, HDAC1 acts to deacetylate H3 and dampens transcription of cytokines.

FIGURE 2

Pharmacological and genetic inhibition of HDAC1 rescue the 2-AA-mediated immunomodulation. (A) Schematic representation of the 2-AA treatments. The human monocytes THP-1 cells and mouse macrophage RAW264.7 cell cells were left untreated (No Pre: No Tolerization) or pretreated with 2-AA for 24 h or 48 h (2-AA Pre: 2-AA tolerization) respectively, and then stimulated (Sti) with 2-AA. (B) Expression of TNF (1 h) was measured in non-pretreated, 2-AA pretreated and 2-AA + TSA pretreated THP-1 cells following 2-AA stimulation. Transcript levels were assessed by qRT-PCR and normalized to GAPDH. (C) ChIP assay of H3K18ac at the TNF promoter of 2-AA-pretreated, 2-AA and TSA pretreated or non-pretreated THP-1 cells following 3 h 2-AA stimulation, assessed by qRT-PCR with primer covering the promoter site region of TNF relative to GAPDH. (D,E) ELISA of IL-1β and Mcp1 secretion in culture supernatant of 2-AA pretreated vector control RAW264.7 and HDAC1 KD cells following 6 h 2-AA stimulation (n = 3; means ± SDs; p < 0.05, Student’s t-test).

FIGURE 3

2-AA tolerization decreases the acetylation of NF-κBp65. Immunoblot showing decreased NF-κBp65K310 acetylation in 2-AA-tolerized RAW264.7 cells (A). HDAC1 KD restored NF-κBp65K310 acetylation in 2-AA-tolerized cells following 2-AA stimulation (B). Data are representative of three independent experiments. An asterisk (^∗) indicates significant difference from untreated cells (P < 0.05). DU, densitometry units, bars represent SD.

FIGURE 4

2-AA modulates CBP/p300-mediated HAT activity and the acetylation of CBP/p300. (A) HAT activity in nuclear lysate of 2-AA stimulated and 2-AA-tolerized THP-1 cells following 1 h 2-AA stimulation +/– of C646 (n = 3; means ± SDs; p < 0.05, Student’s t-test). (B) Immunoblot showing decreased CBP/p300 acetylation in 2-AA-tolerized THP-1 cells. Data are representative of three independent experiments.

FIGURE 5

2-AA modulates H3K18 acetylation. Screening for Histone H3 acetylation following 2-AA stimulation. Nuclear extraction was prepared from THP-1 cells 1 h post-stimulation with 2-AA. The interaction of the enzyme in the sample solution with the acetylated H3K peptides was measured by using Histone H3 Peptide Array ELISA kit (number of replicates = 2; means ± SDs).

FIGURE 6

2-AA tolerization induces interaction between NF-κB p50 and HDAC1 and disrupts p65-CBP/p300 binding. (A) Co-IP assay showing p50/HDAC1 interaction in 2-AA stimulated and 2-AA-tolerized THP-1 cells following at 1 h 2-AA stimulation (top blot, far right band). (B,C). Co-IP assay confirming that 2-AA tolerization inhibits the interaction of p65 with CBP and p300 in vector control RAW264.7 cells (B), and demonstrating an increased interaction of p300-p65 in HDAC1 KD cells (C) following 1 h 2-AA stimulation (1 h). WB, Western blot; Co-IP, co-immunoprecipitation. Data are representative of three independent experiments.