HDAC7 is an immunometabolic switch triaging danger signals for engagement of antimicrobial versus inflammatory responses in macrophages

Abstract

The immune system must be able to respond to a myriad of different threats, each requiring a distinct type of response. Here, we demonstrate that the cytoplasmic lysine deacetylase HDAC7 in macrophages is a metabolic switch that triages danger signals to enable the most appropriate immune response. Lipopolysaccharide (LPS) and soluble signals indicating distal or far-away danger trigger HDAC7-dependent glycolysis and proinflammatory IL-1β production. In contrast, HDAC7 initiates the pentose phosphate pathway (PPP) for NADPH and reactive oxygen species (ROS) production in response to the more proximal threat of nearby bacteria, as exemplified by studies on uropathogenic Escherichia coli (UPEC). HDAC7-mediated PPP engagement via 6-phosphogluconate dehydrogenase (6PGD) generates NADPH for antimicrobial ROS production, as well as D-ribulose-5-phosphate (RL5P) that both synergizes with ROS for UPEC killing and suppresses selective inflammatory responses. This dual functionality of the HDAC7-6PGD-RL5P axis prioritizes responses to proximal threats. Our findings thus reveal that the PPP metabolite RL5P has both antimicrobial and immunomodulatory activities and that engagement of enzymes in catabolic versus anabolic metabolic pathways triages responses to different types of danger for generation of inflammatory versus antimicrobial responses, respectively.

Keywords: immunometabolism; inflammation; macrophages; pentose phosphate pathway; uropathogenic Escherichia coli.

Fig. 1.

HDAC7 discriminates between distal and proximal threats during macrophage activation. (A) Model of immunometabolic control of macrophage responses to distal versus proximal danger signals. (B and C) IL-1β and TNF release from HMDM pretreated with TMP195 (10 μM) for 1 h followed by LPS (100 ng/mL), EC958 (MOI 100), or LPS and latex beads for 4 h. Additional nigericin (5 μg/mL) treatment for 2 h followed, where indicated. (D) Immunoblots for pro- and cleaved-IL-1β in indicated BMM populations treated with either LPS (1 ng/mL) or infected with EC958 (MOI 10) for 4 h, followed by nigericin for 1 h (representative of four independent experiments). (E and F) IL-1β release from the indicated BMM populations treated with either LPS (0.5 ng/mL; Left, E and F), infected with EC958 (MOI 10; E, Right), or LPS-coated latex beads (F, Right) for 4 h, followed by nigericin for 1 h. (G) A heatmap showing the expression patterns of pentose phosphate pathway metabolites (highlighted in red) in lysates from wild-type (Hdac7^+/+) and Hdac7^−/− BMM infected with EC958 (MOI 100, 30 min and 2 h). * and ** indicate statistical comparison between Hdac7^+/+ and Hdac7^−/− BMM at 30 min postinfection, and † indicates statistical comparison between Hdac7^+/+ and Hdac7^−/− BMM at 2 h postinfection. Graphical data (mean ± SEM, n = 3 to 7) are combined from three to seven independent experiments (or donors) performed in experimental duplicate. Data are normalized to the Hdac7^+/+ LPS-treated, EC958-infected, or LPS-coated latex beads (E and F). Statistical significance was determined using repeated measure two-way ANOVA followed by Sidak’s multiple comparison test (ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; †P < 0.05).

Fig. 2.

HDAC7 is required for macrophage-mediated Escherichia coli uptake and clearance. (A) Relative intramacrophage bacterial loads at 2 h p.i. in BMM infected with UPEC strain EC958. (B) Relative intramacrophage loads of EC958 at 2 h p.i. in BMM pretreated with the indicated concentrations of TMP195 for 1 h prior to infection. (C) Relative intramacrophage loads of EC958 at 2 h p.i. in BMM either pretreated with TMP195 for 1 h prior to infection or 1 h p.i. (D) Phagocytic uptake of pHrodo Escherichia coli in indicated BMM populations. (E) Phagocytic uptake of pHrodo Escherichia coli in BMM pretreated with the indicated concentrations of TMP195 for 30 min prior to treatment with pHrodo Escherichia coli. Representative flow cytometry plot for BMM (Left) as well as relative median fluorescence intensity (MFI) data (Right) are shown. (F) Mac-Hdac7 BMM were spin-infected with MG1655_mCherry for 5 min, after which cells were fixed at the indicated time points. Immunofluorescence was performed to detect cells harboring fluorescent bacteria. The indicated image (Left) is representative of three independent experiments. Manual quantification of bacterial numbers (Right) was performed across multiple images (minimum 26 cells per time point, per experiment) utilizing Image J. Graphical data (mean ± SEM, n = 3 to 7) are combined from three to seven independent experiments performed in experimental duplicates and are normalized to the Hdac7^+/+ (A and D) or the no TMP195 control sample (B, C, and E). Statistical significance was determined using unpaired nonparametric Mann–Whitney test (A), Kruskal–Wallis test followed by Dunn’s multiple comparison test (B, C, and E) or repeated measure two-way ANOVA (D and F), followed by Sidak’s multiple comparison test or (ns, not significant; *P < 0.05; ****P < 0.0001).

Fig. 3.

HDAC7 is required for host defense in vivo. (A) Schematic representation of infection strategy. (B–E) Core body temperature (B) and cfu in peritoneal cavity (C), liver (D), and spleen (E) of C57BL/6J mice infected with EC958 in the presence or absence of TMP195. (F–I) Core body temperature (F) and cfu in peritoneal cavity (G), liver (H) and spleen (I) of Hdac7^+/+ and Hdac7^−/− mice infected with EC958. Data show median ± interquartile range of 10 mice per treatment group. Statistical significance was determined by two-way ANOVA (B and F) or unpaired nonparametric Mann–Whitney test (C–E and G–I) (*P < 0.05; **P < 0.01; ***P < 0.001).

Fig. 4.

Antimicrobial effects of HDAC7 require PPP- and NADPH oxidase-dependent ROS. (A) NADPH/NADP⁺ ratio in the indicated BMM populations infected with EC958. (B) Measurement of real time OCR in the indicated BMM populations infected with MG1655 (Left). Area under the curve analysis until 15 min of OCR determinations was calculated with post-background correction (Right). (C–E) Measurement of total ROS in the indicated BMM populations (C and D) or RAW264.7 (E), pretreated with cM-H₂DCFDA (10 μM) for 30 min before infecting them with MG1655 for 30 min. Representative flow cytometry plot (Left), as well as relative median fluorescence intensity (MFI) data (Right), are shown for (C and D). (F) Relative intramacrophage EC958 loads at 2 h p.i. in indicated BMM populations, pretreated with the NOX2 inhibitor GSK2795039 (GSK, 10 μM) for 1 h. (G) Relative intramacrophage bacterial loads of EC958 or EC958katG at 2 h p.i. in the indicated BMM populations. Graphical data (mean ± SEM, n = 3) are combined from three independent experiments and are normalized to the Hdac7^+/+ uninfected control (C), uninfected MacBlue control (D), uninfected empty vector control (E), or MacBlue control infected with wild type EC958 (F and G). Statistical significance was determined using repeated measure two-way ANOVA followed by Sidak’s multiple comparison test [A and B (Left)–G] or unpaired t test with Welch’s correction [B (Right)] (ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Fig. 5.

HDAC7 interacts with and activates 6PGD. (A) 6PGD activity assay on lysates from BMM that had been treated for 0.5 h or 2 h with either LPS (1 ng/mL), latex beads, or LPS plus latex beads or infected with EC958 (MOI 100). (B) Immunoblot of coimmunoprecipitation assay using HEK293T lysates to assess 6PGD interactions (representative of three independent experiments). (C) AlphaLISA assay to analyze the interaction between HDAC7 and 6PGD, PKM2, or FKBP (negative control) following cell-free expression of indicated proteins. (D) Immunoblot (representative of three independent experiments, Left) of coimmunoprecipitation assay using HEK293T lysates to assess strength of HDAC7 interaction with target proteins (6PGD versus PKM2). Interactions were quantified by densitometric analysis of FLAG-tagged proteins (Right). (E) 6PGD activity assay on lysates from the indicated BMM populations infected with EC958 for 2 h. (F and G) 6PGD activity assay on lysates from BMM (F) or HMDM (G) pretreated with TMP195 (10 μM) for 1 h followed by infection with EC958 for 2 h. (H and I) 6PGD activity assay on lysates from HEK293T cells cotransfected with the indicated constructs (H). Immunoblotting was performed on the lysates to confirm similar expression of the proteins (I, representative of three independent experiments). Graphical data (mean ± SEM, n = 3 to 4) are combined from at least three independent experiments and are normalized to untreated control (A), Hdac7^+/+ uninfected control (E), uninfected control (F), or 6PGD alone (H). 6PGD enzyme activity was normalized to the total protein in the lysate (A, E–G). Statistical significance was determined using two-way ANOVA (A and C–E), repeated measure one-way ANOVA with Giesser Greenhouse correction (F and H), or nonparametric Kruskal–Wallis test (G) followed by Tukey’s, Dunn’s, Dunnett’s, or Sidak’s multiple comparison test (ns, not significant; *P < 0.05; **P < 0.01; ****P < 0.0001).

Fig. 6.

HDAC7 acts via the PPP metabolite RL5P to prioritize responses to proximal danger signals. (A) IL-1β release from the indicated BMM populations infected with either EC958 (MOI 10) or treated with LPS (100 ng/mL) for 4 h, followed by an additional treatment with nigericin for 1 h. (B) IL-1β release from the indicated BMM populations pretreated with the PPP inhibitors 6-aminonicotinamide (6-AN) (0.5 μM) or polydatin (PD) (0.5 μM) for 1 h, followed by infection with EC958 for 4 h. Additional nigericin treatment for 1 h followed. (C) IL-1β release from BMM knocked down for the indicated genes, treated with LPS (0.5 ng/mL) for 4 h followed by nigericin treatment for 1 h. (D) IL-1β release from BMM retrovirally transduced with indicated constructs, treated with LPS (0.5 ng/mL) for 4 h followed by an additional treatment with nigericin for 1 h. (E–H) IL-1β and TNF release from HMDM and BMM pretreated with the indicated concentrations of ribulose-5-phosphate for 16 h, followed by LPS (10 ng/mL) or infection with EC958 for 4 h. Additional treatment with nigericin for 1 h followed. (I and J) Growth curve analysis (A₆₀₀) of EC958 cultured ± H₂O₂ (0.8 mM) in the presence of the indicated concentrations of RL5P (I) or 6-phosphogluconate (6PG) (J) for 12 h. All graphical data (mean ± SEM, n = 3 to 6) are combined from three to six independent experiments unless otherwise specified and are normalized to the MacBlue (A), Hdac7^+/+ (B), LPS-treated no siRNA control (C), LPS-treated empty vector control (D), or LPS-treated BMMs (F, H). Statistical significance was determined using repeated measure two-way ANOVA followed by Sidak’s, or Dunnett’s multiple comparison test (A–H) (ns, not significant; *P < 0.05; **P < 0.01; ****P < 0.0001).