O6-methylguanine DNA methyltransferase regulates β-glucan-induced trained immunity of macrophages via farnesoid X receptor and AMPK

Summary: Trained immunity is the heightened state of innate immune memory that enhances immune response resulting in nonspecific protection. Epigenetic changes and metabolic reprogramming are critical steps that regulate trained immunity. In this study, we reported the involvement of O6-methylguanine DNA methyltransferase (MGMT), a DNA repair enzyme of lesion induced by alkylating agents, in regulation the trained immunity induced by β-glucan (BG). Pharmacological inhibition or silencing of MGMT expression altered LPS stimulated pro-inflammatory cytokine productions in BG-trained bone marrow derived macrophages (BMMs). Targeted deletion of Mgmt in BMMs resulted in reduction of the trained responses both in vitro and in vivo models. The transcriptomic analysis revealed that the dampening trained immunity in MGMT KO BMMs is partially mediated by ATM/FXR/AMPK axis affecting the MAPK/mTOR/HIF1α pathways and the reduction in glycolysis function. Taken together, a failure to resolve a DNA damage may have consequences for innate immune memory

Notch signaling regulates the responses of lipopolysaccharide-stimulated macrophages in the presence of immune complexes

<div><p>Macrophages exhibit diverse effector phenotypes depending on the stimuli and their microenvironment. Classically activated macrophages are primed with interferon (IFN)γ and stimulated with pathogen-associated molecular patterns. They produce inflammatory mediators and inflammatory cytokines, such as IL-12. In the presence of immune complexes (ICs), activated macrophages have decreased IL-12 production and increased IL-10 production and presumably act as regulatory macrophages. Notch signaling has been shown to regulate the effector functions of classically activated macrophages. In this study, we investigated whether Notch signaling is active in lipopolysaccharide (LPS)-stimulated macrophages in the presence of ICs. LPS/IC stimulation increased the level of cleaved Notch1 in murine macrophages, while IC stimulation alone did not. Delta-like 4, but not Jagged1, was responsible for generating cleaved Notch1. The activation of Notch signaling by LPS/ICs depended upon NF-κB and MEK/Erk pathway activation. Macrophages with the targeted deletion of <i>Rbpj</i>, which encodes a DNA-binding protein central to canonical Notch signaling, produced significantly less IL-10 upon LPS/IC stimulation. A similar impact on IL-10 production was observed when Notch signaling was inhibited with a gamma-secretase inhibitor (GSI). Defects in NF-κB p50 nuclear localization were observed in GSI-treated macrophages and in <i>Rbpj</i>^<i>-/-</i> macrophages, suggesting cross-regulation between the Notch and NF-κB pathways. Transcriptomic analysis revealed that Notch signaling regulates the transcription of genes involved in the cell cycle, macrophage activation, leukocyte migration and cytokine production in LPS/IC-stimulated macrophages. Taken together, these results suggest that the Notch signaling pathway plays an important role in regulating the functions of macrophages activated by LPS and ICs.</p></div

Defects in NF-κB p50 nuclei accumulation by GSI treatment or deletion of CSL/RBP-Jκ in LPS/IC-activated macrophages.

<p>(A) BMMs were pretreated with DMSO or GSI for 1 and 4 hr and the cytosolic and nuclei fractions were analyzed for NF-κB p50 by Western blotting. GAPDH and CSL/RBP-Jκ were used as cytosolic and nuclei markers, respectively. (B) BMMs from wildtype or <i>Rbpj</i> KO mice were stimulated with LPS/IC for 1 and 4 hr and the cytosolic and nuclei fractions were analyzed for NF-κB p50 as described for (A). Representative data from 1 of 2 independent experiments are shown.</p

Effects of inhibiting Notch signaling by GSI or targeted <i>Rbpj</i> deletion on IL-10 production in LPS/IC-activated macrophages.

<p>(A) IFNγ-primed BMMs were pre-treated with vehicle control DMSO or GSI (25 μM) for 30 min. The cells were subsequently activated by LPS/ICs for 5, 15, 30 and 60 min. Cleaved Notch1 (Val 1744) and Notch1 were measured in whole cell lysates by Western blotting. Representative data from one of two independent experiments are shown. (B) IFNγ-primed BMMs were pre-treated with vehicle control DMSO or GSI (25 μM) and subsequently activated by LPS/ICs for 6 hrs. IL-10 expression was detected by intracellular cytokine staining. Representative data from one of two independent experiments are shown. (C) Culture supernatants were collected from BMMs stimulated for 6 hrs as described in (B), and the IL-10 levels were measured by ELISA. (D) CSL/RBP-Jκ expression in BMMs from wildtype (ctrl) or <i>Rbpj</i> KO (CSL KO) mice was detected by Western blotting. Representative data from one of two independent experiments are shown. BMMs from wildtype (ctrl) or <i>Rbpj</i> KO mice were activated by LPS or LPS/ICs for 6 hrs. IL-10 levels were detected by ELISA. * and ** indicate statistical significance (<i>p</i><0.05) according to one-way ANOVA with Tukey’s multiple comparison test. The results represent the mean±SD of triplicate determinations from one of two independent experiments.</p

qPCR validation of differential gene expression in wildtype or <i>Rbpj</i> KO macrophages.

<p>Representative genes whose levels of expression were downregulated (A) or upregulated (B) by GSI treatment were validated in BMMs from wildtype or <i>Rbpj</i> KO mice upon activation by LPS/ICs. *, ** and *** indicate statistical significance (<i>p</i><0.05) according to unpaired t-tests. The data represent the mean±SD of triplicate determinations from one of two representative independent experiments.</p

Effect of specific inhibitors of the MAPK, NF-κB and PI3K signaling pathways on Notch signaling activation and IL-10 production in LPS/IC-activated macrophages.

<p>(A) IFNγ-primed BMMs were pre-treated with Bay-11 (10 μM), SB203580 (10 μM), U0126 (10 μM), LY94002 (50 μM) or vehicle control DMSO for 30 min. The cells were subsequently activated by LPS/ICs for 1 hr, and protein lysates were created. Notch1 and cleaved Notch1 (Val 1744) were detected by Western blot. β-actin was used as the loading control. Representative data from one representative experiment of 3 independent experiments are shown. (B) Culture supernatants from the cells treated as in (A) were collected 6 hrs after stimulation, and the IL-10 levels were measured by ELISA. ** indicates statistical significance (<i>p</i><0.05) according to one-way ANOVA with Tukey’s multiple comparison test. The results represent the mean±SD of triplicate determinations from one representative experiment of 2 independent experiments.</p

Cytokine expression in macrophages stimulated with LPS or LPS/ICs.

<p>(A) IFNγ-primed BMMs were activated by LPS (100 ng/mL) or LPS/ICs for 4 hrs. The expression levels of <i>Il10</i> and <i>Il12b</i> mRNA were measured by qPCR. (B-D) IFNγ-primed BMMs were activated by ICs (anti-OVA IgG) in the presence or absence of LPS (100 ng/mL) for 6 hrs. The levels of IL-10 (B), IL-6 (C) and TNFα (D) in the culture supernatant were measured by ELISA. ** indicates statistical significance (<i>p</i><0.05) according to one-way ANOVA with Tukey’s multiple comparison test. n.s. indicates no statistical significance. The results represent the mean±SD of triplicate determinations from one representative experiment of two independent experiments.</p

qPCR validation of differential gene expression induced by GSI treatment.

<p>Representative genes whose levels of expression were downregulated (A) or upregulated (B) by GSI treatment were validated in IFNγ-primed BMMs activated by LPS/ICs and treated with vehicle control DMSO or GSI. *, ** and *** indicate statistical significance (<i>p</i><0.05) according to unpaired t-tests. The data represent the mean±SD of triplicate determinations from 1 of 2 representative independent experiments.</p

Effects of GSI treatment on MAPK, PI3K/AKT and NF-κB pathway activation in LPS/IC-activated macrophages.

<p>(A-B) IFNγ-primed BMMs were pre-treated with vehicle control DMSO or GSI (25 μM) for 30 min and activated by LPS/ICs for 5, 15, 30 and 60 min. Phospho-p38, p38 phospho-p44-42, p44-42, phospho-SAPK/JNK, SAPK/JNK, phospho-Akt, Akt and the loading control β-actin were detected by Western blotting. Representative data from 1 of 3 independent experiments are shown. (C) IFNγ-primed BMMs were activated by LPS/ICs for 4 hrs in the presence of vehicle control DMSO or GSI (25 μM). NF-κB p50 was detected by immunofluorescence staining. The arrows indicate cells with decreased or no p50 nuclear translocation (green). Representative data from 1 of 2 independent experiments are shown.</p