The Interplay of Notch Signaling and STAT3 in TLR-Activated Human Primary Monocytes

Abstract

The highly conserved Notch signaling pathway essentially participates in immunity through regulation of developmental processes and immune cell activity. In the adaptive immune system, the impact of the Notch cascade in T and B differentiation is well studied. In contrast, the function, and regulation of Notch signaling in the myeloid lineage during infection is poorly understood. Here we show that TLR signaling, triggered through LPS stimulation or in vitro infection with various Gram-negative and -positive bacteria, stimulates Notch receptor ligand Delta-like 1 (DLL1) expression and Notch signaling in human blood-derived monocytes. TLR activation induces DLL1 indirectly, through stimulated cytokine expression and autocrine cytokine receptor-mediated signal transducer and activator of transcription 3 (STAT3). Furthermore, we reveal a positive feedback loop between Notch signaling and Janus kinase (JAK)/STAT3 pathway during in vitro infection that involves Notch-boosted IL-6. Inhibition of Notch signaling by γ-secretase inhibitor DAPT impairs TLR4-stimulated accumulation of NF-κB subunits p65 in the nucleus and subsequently reduces LPS- and infection-mediated IL-6 production. The reduced IL-6 release correlates with a diminished STAT3 phosphorylation and reduced expression of STAT3-dependent target gene programmed death-ligand 1 (PD-L1). Corroborating recombinant soluble DLL1 and Notch activator oxaliplatin stimulate STAT3 phosphorylation and expression of immune-suppressive PD-L1. Therefore we propose a bidirectional interaction between Notch signaling and STAT3 that stabilizes activation of the transcription factor and supports STAT3-dependent remodeling of myeloid cells toward an immuno-suppressive phenotype. In summary, the study provides new insights into the complex network of Notch regulation in myeloid cells during in vitro infection. Moreover, it points to a participation of Notch in stabilizing TLR-mediated STAT3 activation and STAT3-mediated modulation of myeloid functional phenotype through induction of immune-suppressive PD-L1.

Keywords: DLL1; Notch signaling; PD-L1; STAT3; TLR; infection.

Figure 1

TLR signaling induces DLL1 in primary human monocytes. CD14⁺ monocytes, isolated from blood of healthy donors were stimulated with 100 ng/ml LPS or infected with Escherichia (E.) coli, Enterococcus (E.) faecalis, Klesiella (K.) pneumoniae, Pseudomonas (P.) aeruginosa in a concentration of 10⁶ bacteria per 10⁶ monocytes/ml. After 2 h bacteria were killed by gentamicin. The next day cells and supernatant were analyzed. (A) RNA was isolated and cDNA produced. Induction of gene expression was analyzed by qRT PCR using sequence-specific primer for DLL1 and JAG1 (gene encoding Jagged-1) and SYBR Green Master mix. Actin was detected as endogenous control for normalization. (B) For western blot analysis equal amounts of protein lysates were blotted and probed with antibodies against DLL1 or GAPDH (loading control). Shown is one representative blot and the associated quantification. Quantification was performed using the Image Analysis System Bioprofil (Fröbel, Germany). The intensity of signals were calculated against loading control and presented as percent of untreated samples. (C) Supernatants were used for ELISA analysis to quantify shedded DLL1 extracellular domain. (A,C) depict the mean and standard deviation of at least three donors. Statistics: ^*p ≤ 0.05 by Mann–Whitney U-test.

Figure 2

TLR activation induces Notch signaling. Primary human monocytes were treated as described in Figure 1. (A) HES and HEY gene expression was analyzed by qRT PCRs. (B) Protein expression was detected by western blot analyses. Results were quantified. (A) Shows the mean and standard deviation of three donors. Statistics: ^*p ≤ = 0.05 by Mann–Whitney U-test.

Figure 3

LPS-induced systemic inflammation in mice. Twelve week old male mice were injected intraperitoneally with LPS or NaCL (control group). After 24 h blood were taken and analyzed for DLL1 by ELISA analyses. Statistics: ^*p ≤ = 0.05 by Mann–Whitney U-test.

Figure 4

TLR4-signaling promotes DLL1 expression through STAT3. Human blood-derived monocytes were pretreated with STAT3 Inhibitor JSI-124 (200 nM) for 2 h before stimulation with LPS or infection. (A) The next day lysates were produced and applied for western blot analyses for the detection of phosphorylated (Tyr 701) STAT3 and DLL1. GAPDH was detected as loading control. The experiment was repeated two times with comparable results. (B) Quantification of (A). (C) Supernatant was analyzed by ELISA for shedded DLL1. (D) JSI-124 pretreated and E. coli-infected cells were stimulated with 0.5 or 1 μM oxaliplatin (oxa). The next day cDNA was produced and analyzed for HES and HEY induction. (C,D) mean ± std n = 3, ^*p ≤ = 0.05 by Mann–Whitney U-test.

Figure 5

Notch signaling augments TLR4-stimulated IL-6 and TNFα expression. Monocytes were pretreated with DAPT (2.5 μM) for 1 h before LPS stimulation and E. coli infection. 2 h after infection, medium was changed and DAPT was added again. (A) The next day RNA was isolated, cDNA produced and gene expression was analyzed by qRT PCR using sequence-specific primer for HES and Hey and SYBR Green. Results were normalized against actin. (B) Supernatants were used for ELISA analysis to quantify released IL-6, IL-12p40, and TNFα. (C) For western blot analysis cells were harvested 2 h after infection. Nuclear lysates were produced and equal amounts of lysates were blotted and probed with antibodies against p65 or B23 (control). Shown is one representative experiment out of three and the associated quantification of three experiments. (A–C) mean ± std n = 3, ^*p ≤ = 0.05 by Mann–Whitney U-test.

Figure 6

Notch signaling modulates functional myeloid phenotype. Primary blood-derived monocytes were stimulated with 3 μg/ml recombinant soluble DLL1 ± 1 μM oxaliplatin (oxa). Aside from that cells were treated with DAPT (1 μM) 1 h before infection with E. coli. (A) Cell lysates were analyzed by western blots for STAT3 phosphorylation. GAPDH was detected as loading control. Results were quantified. (B) Monocytes were treated with an anti-IL6 specific antibody (Thermo Fisher Scientific) in parallel to LPS (100 ng/ml) stimulation. The next day lysates were produced and used in western blot analyses for detection of p(y701)STAT3 and DLL1. Loading control: GAPDH. Experiment was repeated two times with comparable results. (C) After 3 days cells were analyzed with fluorescently-labeled antibodies by flow cytometry for surface expression of PD-L1. Shown are FACS histogram overlays (Weasel.jar software) and the associated quantification of three experiments. Mean ± std n = 3, ^*p ≤ = 0.05 by Mann–Whitney U-test.

Figure 7

Positive feedback loop between Notch signaling and STAT3 after TLR-activation. (1+1A) LPS stimulates IL-6 production through TLR4-mediated NF-κB signaling. (2) IL-6 binds to IL-6 receptor (IL-6R) and stimulates activation of STAT3. (3) STAT3 induces expression of DLL1. (4) Transmembrane DLL1 binds to Notch receptor on neighboring cells. Notch receptor intracellular domain (NICD) translocates to the nucleus, induces HES/HEY transcription, and mediates NF-κB accumulation in the nucleus. IL-6 production is enhanced. (5) Enhanced IL-6 receptor signaling stabilizes STAT3 activation. (6) STAT3-dependent PD-L1 expression is increased.