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. 2023 Nov 16;12(11):1441.
doi: 10.3390/biology12111441.

Prostaglandin E2 Boosts the Hyaluronan-Mediated Increase in Inflammatory Response to Lipopolysaccharide by Enhancing Lyve1 Expression

Pauline Hog  1 , Silvia Kuntschar  1 , Peter Rappl  1 , Arnaud Huard  1 , Andreas Weigert  1   2   3 , Bernhard Brüne  1   2   3   4 , Tobias Schmid  1   2
Affiliations

Prostaglandin E2 Boosts the Hyaluronan-Mediated Increase in Inflammatory Response to Lipopolysaccharide by Enhancing Lyve1 Expression

Pauline Hog et al. Biology (Basel). .

Abstract

Macrophages are a highly versatile and heterogenic group of immune cells, known for their involvement in inflammatory reactions. However, our knowledge about distinct subpopulations of macrophages and their specific contribution to the resolution of inflammation remains incomplete. We have previously shown, in an in vivo peritonitis model, that inhibition of the synthesis of the pro-inflammatory lipid mediator prostaglandin E2 (PGE2) attenuates efficient resolution of inflammation. PGE2 levels during later stages of the inflammatory process further correlate with expression of the hyaluronan (HA) receptor Lyve1 in peritoneal macrophages. In the present study, we therefore aimed to understand if PGE2 might contribute to the regulation of Lyve1 and how this might impact inflammatory responses. In line with our in vivo findings, PGE2 synergized with dexamethasone to enhance Lyve1 expression in bone marrow-derived macrophages, while expression of the predominant hyaluronan receptor CD44 remained unaltered. PGE2-mediated Lyve1 upregulation was strictly dependent on PGE2 receptor EP2 signaling. While PGE2/dexamethasone-treated macrophages, despite their enhanced Lyve1 expression, did not show inflammatory responses upon stimulation with low (LMW) or high-molecular-weight hyaluronan (HMW)-HA, they were sensitized towards LMW-HA-dependent augmentation of lipopolysaccharide (LPS)-induced inflammatory responses. Thus, Lyve1-expressing macrophages emerged as a subpopulation of macrophages integrating inflammatory stimuli with extracellular matrix-derived signals.

Keywords: hyaluronic acid; inflammation; macrophage; peritonitis; resolution.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
mPGES-1 inhibition attenuates Lyve1 expression in a zymosan-induced peritonitis model. (A) Peritonitis was induced in C57/BL6 mice by i.p. injection of 5 mg/kg zymosan (Z). Starting 24 h after zymosan injection, the mPGES-1 inhibitor CIII (25 mg/kg) or the appropriate vehicle control were applied i.p. daily, to study the role of PGE2 in the resolution of inflammation. (B) Lyve1 and (C) CD44 mRNA expression in macrophages (MΦ) isolated from the peritoneum was assessed by RNA-seq at days 0, 1, 3, and 6. Data are given as mean library-normalized read counts relative to day 0. (D) The percentage of MΦ presenting Lyve1 at the surface relative to all MΦ (F4/80hi, MERTKhi) was determined by FACS analysis at day 6. Data are presented as mean ± SEM (n > 7; * p < 0.05).
Figure 2
Figure 2
PGE2 enhances dexamethasone-induced Lyve1 expression. Bone marrow-derived macrophages (BMDM) were differentiated for 5 days before stimulation for 48 h with (A) dexamethasone (dexa; 100 ng/mL), PGE2 (250 ng/mL), or a combination of dexa and PGE2. (B) To determine the role of the PGE2 receptors EP1-4, BMDM were separately pre-incubated with selective antagonists for EP1 (ONO 8130), EP2 (PF-04418948), EP3 (L-798,106), or EP4 (ONO AE3 208) (1 µM each) 30 min before stimulation with dexa and PGE2 for 48 h. mRNA expression of Lyve1 (upper panels) and CD44 (lower panels) was determined by RT-qPCR analysis. Data are normalized to Tbp (TATA-box binding protein) and presented relative to corresponding controls as mean ± SEM (n > 12; * p < 0.05; ** p < 0.01; *** p < 0.001; compared to respective controls (* untreated, # dexa, § PGE2, or & dexa + PGE2 stimulated)).
Figure 3
Figure 3
Hyaluronan amplifies LPS-induced inflammatory responses only in PGE2/dexamethasone-primed macrophages. Bone marrow-derived macrophages (BMDM) were differentiated for 5 days, primed for 48 h with dexamethasone (dexa; 100 ng/mL) and PGE2 (250 ng/mL), and treated for 1 h with high-molecular-weight hyaluronan (HMW-HA) (500 µg/mL or 1000 µg/mL) (A) or low-molecular-weight hyaluronan (LMW-HA) (500 ng/mL or 500 µg/mL) (B) prior to inflammatory stimulation with lipopolysaccharide (LPS; 100 ng/mL) for 1 h. mRNA expression of Tnf (upper panels) and Il10 (lower panels) was determined by RT-qPCR analysis. Data are normalized to Tbp and presented relative to the untreated control as mean ± SEM (n = 5; * p < 0.05; ** p < 0.01; *** p < 0.001; compared to respective controls (* untreated, # dexa + PGE2, or § dexa + PGE2 + LPS stimulated).
Figure 4
Figure 4
PGE2/dexamethasone priming sensitizes macrophages to enhanced inflammatory responses by hyaluronan via EP2 receptor. (A) Bone marrow-derived macrophages (BMDM) differentiated for 5 days, were pre-treated with the EP2 antagonist PF-04418948 (1 µM) for 30 min before priming for 48 h with dexamethasone (dexa; 100 ng/mL) and PGE2 (250 ng/mL). Then, the cells were treated for 1 h with low-molecular-weight hyaluronan (LMW-HA) (500 µg/mL) prior to inflammatory stimulation with lipopolysaccharide (LPS; 100 ng/mL) for 1 h. mRNA expression of Tnf was determined by RT-qPCR analysis. Data are normalized to Tbp and presented relative to LPS + LMW-HA-treated cells as mean ± SEM (n > 8; * p < 0.05; ** p < 0.01; compared to the respective LPS + LMW-HA-stimulated control (* no treatment, # dexa + PGE2 treatment)). (B) Schematic model of the proposed mechanism of PGE2-dependent aggravated inflammatory responses in macrophages by enhanced sensitization to LMW-HA via Lyve1.
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