A quorum-sensing signal promotes host tolerance training through HDAC1-mediated epigenetic reprogramming

Abstract

The mechanisms by which pathogens evade elimination without affecting host fitness are not well understood. For the pathogen Pseudomonas aeruginosa, this evasion appears to be triggered by excretion of the quorum-sensing molecule 2-aminoacetophenone, which dampens host immune responses and modulates host metabolism, thereby enabling the bacteria to persist at a high burden level. Here, we examined how 2-aminoacetophenone trains host tissues to become tolerant to a high bacterial burden, without compromising host fitness. We found that 2-aminoacetophenone regulates histone deacetylase 1 expression and activity, resulting in hypo-acetylation of lysine 18 of histone H3 at pro-inflammatory cytokine loci. Specifically, 2-aminoacetophenone induced reprogramming of immune cells occurs via alterations in histone acetylation of immune cytokines in vivo and in vitro. This host epigenetic reprograming, which was maintained for up to 7 days, dampened host responses to subsequent exposure to 2-aminoacetophenone or other unrelated pathogen-associated molecules. The process was found to involve a distinct molecular mechanism of host chromatin regulation. Inhibition of histone deacetylase 1 prevented the immunomodulatory effects of 2-aminoacetophenone. These observations provide the first mechanistic example of a quorum-sensing molecule regulating a host epigenome to enable tolerance of infection. These insights have enormous potential for developing preventive treatments against bacterial infections.

Figure 1. 2-AA pretreatment modulates activation of NF-κB and cytokines, and promotes long-term immunosuppression

(a) Experimental design: Human THP-1 monocytes were left untreated (No Pre) or pretreated with 2AA for 24-h (2-AA Pre), and then stimulated (Sti) with 2-AA. (b) SEAP assay analyzing NF-κB reporter activation in No Pre and 2-AA Pre cells after 2AA stimulation (4-h). (N = 3; means ± SDs; p < 0.05, Student's t test). (c-e) Real-time PCR analysis of Tnf-α (1-h), IL-1β (1-h) and Mcp-1 (3-h) mRNA in No Pre and 2-AA Pre cells following 2AA stimulation. Transcript levels were normalized to β-actin (N = 3; means ± SDs). (f-h) ELISA of pro-inflammatory cytokines in supernatants of No Pre and 2-AA Pre cells following 6-h 2-AA stimulation (N = 3; means ± SDs; p < 0.05, one-way ANOVA). (i-j) ELISA of TNF-α and MCP-1 in supernatants of No Pre and 2-AA Pre primary human macrophages, which were washed out and after 7 days and stimulated with 2-AA, LPS, or PGN for 6-h (N=3, means ± SD, p < 0.05, one-way ANOVA). Data are representative of three independent experiments.

Figure 2. 2-AA pretreatment modulates histone acetylation

(a-b) Global acetylation levels of H3K18 and H3K9 in mouse macrophages in 2-AA pretreated and non-pretreated cells following 2-AA stimulation. (c) Red lines denote promoter 1, promoter 2, and intron primer target at Tnf-α locus. (d-f) ChIP assay of H3K18ac, H3K9ac, and H3K9me3 at the Tnf-α promoter in 2-AA pretreated or non-pretreated RAW264.7 cells following 3-h 2AA stimulation, assessed by real-time PCR with primers covering promoter sites and intronic region (as in c) of Tnf-α (d-e). The intronic region was unaffected (f). (N = 3; means ± SDs; p < 0.05, Student's t test). Data are representative of three independent experiments.

Figure 3. 2-AA pretreatment modulates the HAT/HDAC activity and promotes HDAC1 accumulation

(a) HAT activity in nuclear lysate of 2-AA pretreated THP-1 cells following 1-h 2-AA stimulation (N = 3; means ± SDs; p < 0.05, Student's t test). (b) Nuclear HDAC activity in 2-AA pretreated and non-pretreated THP-1 cells following 2AA stimulation +/−TSA, +/−VPA, +/− Ms275, +/− TMP269, +/− RGFP966, +/−PCI-34051, +/− HPOB or +/− NIC for 6-h, (N = 3; means ± SDs; p < 0.05, one-way ANOVA). (c) Quantitation of nuclear HDAC1 in 2AA-pretreated and nonpretreated THP-1 cells following 3-h 2-AA stimulation. (N = 3; means ± SDs; p < 0.05, Student's t test). (d-e) ChIP assay of HDAC1 abundance at Tnf-α promoter in 2-AA pretreated or non-pretreated RAW264.7 cells following 3-h 2-AA stimulation, assessed by real-time PCR with primers covering H3K18ac enriched site in Tnf-α promoter region (N = 3; means ± SDs; p < 0.05, Student's t test). Data are representative of three independent experiments.

Figure 4. HDAC1 inhibition reinstates histone acetylation and NF-κB activation, and modulates cytokine secretion

(a) Immunoblot analysis of H3K18 acetylation in 2-AA pretreated vector control RAW264.7 and HDAC1 KD cells following 2-AA stimulation. Blots are representative of three independent experiments. (b) ELISA of TNF-α secretion in culture supernatants of 2-AA pretreated and nonpretreated vector control RAW264.7 and HDAC1 KD cells following 2-AA stimulation +/− TSA for 6-h. (c) SEAP assay of NF-κB activation in 2-AA pretreated or non-pretreated vector control RAW264.7 and HDAC1 KD cells following 4-h 2-AA stimulation. (d) ELISA of TNF-α levels in culture supernatants of THP-1 cells pre-exposed (or not) to 2-AA with TSA, VPA, Ms275, or NIC following 6-h 2-AA stimulation. Data are representative of three independent experiments (b-d; N = 3; means ± SDs; p < 0.05, one-way ANOVA)

Figure 5. HDAC1 is important for 2-AA mediated host tolerance training in vivo

(a) Kaplan-Meier survival curves of BI mice infected with the PA clinical isolate PA14 (dotted black, N= 16), BI mice exposed to 2-AA 4 days prior to BI (red, N= 14), BI mice treated with 2-AA 4 days before BI and TSA after BI (dotted green, N = 14), and BI mice treated with TSA following BI (dotted purple, N = 16). Non-BI TSA-only treated mice (dotted yellow, N=10) served as a negative control group. All TSA-treated animals received TSA daily for 10 days. (b) Bacterial burden (as CFUs) in muscle 5 days posttreatment in BI mice that received 2-AA treatment 4 days prior to BI with (green, N = 6) or without TSA (red, N= 7); non-pretreated BI minus TSA (black N = 6), or plus TSA following BI (purple, N = 7). The TSA-only treatment group (yellow, N = 10) served as a negative control group. The significance between the infection groups was assessed by the Kruskal-Wallis test (p < 0.05). Each circle represents an individual mouse; small horizontal lines represent the median of log values. Dotted line represents the detection limit (d.l.). (c) Analysis of global level of H3K18 acetylation in the spleens of naïve, BI only, BI with TSA, 2-AA pretreated BI without TSA treatment, and 2-AA pretreated BI groups 1 day, 5 days, and 10 days post-BI. (d-e) Nuclear HAT (d) and HDAC (e) activity in spleens of 2-AA pretreated and non-pretreated BI mice with or without TSA 1 day, 5 days, and 10 days post-BI. (f-g) ELISA of TNF-α and MCP-1 in serum from 2-AA pretreated and non-pretreated BI mice with or without TSA. TSA-only treatment served as the negative control condition. Data are representative of two independent experiments (c-g, N = 3 mice/group, means ± SDs; p < 0.05, Student's t test). (h) A schematic representation of 2-AA mediated reprogramming of innate immunity. Following cell stimulation, increased HAT activity initiates histone acetylation, potentiating transcription of pro-inflammatory genes; subsequent histone deacetylation prevents transcription. Upon subsequent stimulation, cells remain unresponsive as a result of sustained 2-AA induced deacetylation by HDAC1.