LDL receptor-related protein-1 regulates NFκB and microRNA-155 in macrophages to control the inflammatory response

Abstract

LDL receptor-related protein-1 (LRP1) is an endocytic and cell-signaling receptor. In mice in which LRP1 is deleted in myeloid cells, the response to lipopolysaccharide (LPS) was greatly exacerbated. LRP1 deletion in macrophages in vitro, under the control of tamoxifen-activated Cre-ER(T) fusion protein, robustly increased expression of proinflammatory cytokines and chemokines. In LRP1-expressing macrophages, proinflammatory mediator expression was regulated by LRP1 ligands in a ligand-specific manner. The LRP1 agonists, α2-macroglobulin and tissue-type plasminogen activator, attenuated expression of inflammatory mediators, even in the presence of LPS. The antagonists, receptor-associated protein (RAP) and lactoferrin (LF), and LRP1-specific antibody had the entirely opposite effect, promoting inflammatory mediator expression and mimicking LRP1 deletion. NFκB was rapidly activated in response to RAP and LF and responsible for the initial increase in expression of proinflammatory mediators. RAP and LF also significantly increased expression of microRNA-155 (miR-155) after a lag phase of about 4 h. miR-155 expression reflected, at least in part, activation of secondary cell-signaling pathways downstream of TNFα. Although miR-155 was not involved in the initial induction of cytokine expression in response to LRP1 antagonists, miR-155 was essential for sustaining the proinflammatory response. We conclude that LRP1, NFκB, and miR-155 function as members of a previously unidentified system that has the potential to inhibit or sustain inflammation, depending on the continuum of LRP1 ligands present in the macrophage microenvironment.

Keywords: LDL receptor-related protein-1; NFκB; lipopolysaccharide; microRNA-155; tissue-type plasminogen activator.

Fig. 1.

LPS toxicity is increased in mice in which LRP1 is deleted in myeloid cells. (A) IHC was performed to detect CD11b-positive myeloid cells in sections of lung from untreated mLRP1^+/+ and mLRP1^−/− mice. (Scale bar, 100 µm.) Image analysis did not reveal a difference in the density of CD11b-positive cells (n = 3). (B) Kaplan–Meier survival curves are shown for mLRP1^−/− and mLRP1^+/+ mice treated by i.p. injection with 1 mg/kg LPS or vehicle (Veh). Significance was determined by log-rank test (***P < 0.001). (C and D) ELISAs were performed to detect TNFα and CCL3 in plasma samples harvested at the indicated times from LPS-treated mLRP1^−/− (blue bar) and mLRP1^+/+ (red bar) mice (mean ± SEM; n = 10; *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA with Tukey’s post hoc analysis). (E–H) RNA was harvested from the lungs (E and F) and kidneys (G and H) of mLRP1^+/+ and mLRP1^−/− mice 24 h after i.p. injection of LPS or vehicle (C). IL-6 and iNOS mRNA were determined by RT-qPCR (n = 4).

Fig. 2.

LRP1 gene deletion in macrophages in vitro induces a proinflammatory phenotype. BMDMs from Cre-ER^T-negative (control) and Cre-ER^T-positive (Cre-ERT) LRP1^flox/flox mice were treated with 5 µM TAM (+) or vehicle (−) for 7 d. (A) Cell extracts were subjected to immunoblot analysis to detect the LRP1 β-chain and β-actin as a control for load. (B–F) RNA was isolated and RT-qPCR was performed to quantitate mRNA for (B) TNFα, (C) IL-6, (D) IL-1β, (E) CCL2, and (F) CCL3. (G) Cell migration was studied using Transwell systems. Representative images of cells that migrated to the underside surfaces of the membranes are shown. (H) Quantification of cell migration results (mean ± SEM; n = 6; **P < 0.01, ***P < 0.001; one-way ANOVA with a Tukey’s post hoc analysis).

Fig. S1.

LRP1 gene deletion does not compromise cell viability. BMDMs were isolated from Cre-ER^T-positive-LRP1^flox/flox mice and treated with 5 µM TAM (+TAM) or vehicle (−TAM). (A) Apoptosis was measured using the Cell Death ELISA. (B) Cell viability was determined by trypan blue exclusion (mean ± SEM; n = 4; ns, not statistically significant; Mann–Whitney test).

Fig. 3.

LF and RAP induce expression of proinflammatory cytokines in BMDMs. (A–C) BMDMs from C57BL/6J mice were treated with LPS (0.1 µg/mL), LF (150 nM), RAP expressed as a GST fusion protein (150 nM), purified GST (150 nM), or vehicle (C) for 8 h. RT-qPCR was performed to determine mRNA levels for TNFα, IL-6, and CCL2. (D) CCL3 mRNA was determined in BMDMs treated for 8 h with increasing concentrations of RAP. (E) BMDMs were stimulated for 8 h with LPS, LF, RAP, GST, or vehicle as in A. TNFα in conditioned medium was determined by ELISA. (F) BMDMs were treated with LF, RAP, or vehicle (C). Cell migration was studied using Transwell systems. Representative images of migrated cells are shown. (G) Quantification of cell migration results (n = 6). (H) BMDMs were treated with LF, RAP, GST (each at 150 nM), or vehicle (C) for 8 h. Cell extracts were subjected to immunoblot analysis to detect the LRP1 β-chain and β-actin. (I–K) BMDMs were treated with LRP1-neutralizing antibody (anti-LRP1) or isotype-matched IgG for 8 h. mRNA levels were determined for TNFα, IL-6, and CCL4 (mean ± SEM; n ≥ 6; *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA followed by Dunnett’s multiple comparison test).

Fig. S2.

LF and RAP increase expression of proinflammatory mediators. BMDMs from C57BL/6J mice were treated with LPS (0.1 µg/mL), LF (150 nM), RAP, which was expressed as a GST-fusion protein (150 nM), purified GST (150 nM), or vehicle (C) for 8 h. RT-qPCR was performed to quantify mRNA levels for (A) IL-1β and (B) CCL4 (mean ± SEM; n = 6; **P < 0.01, ***P < 0.001; one-way ANOVA followed by Dunnett’s post hoc analysis).

Fig. 4.

α₂M* and EI-tPA inhibit expression of inflammatory mediators by BMDMs. (A) BMDMs from C57BL/6J mice were treated for 8 h with El-tPA (12 nM), α₂M* (10 nM), or vehicle (C). TNFα mRNA was determined by RT-qPCR. (B–G) BMDMs were pretreated with LPS (0.1 µg/mL) for 30 min and then with El-tPA (12 nM), α₂M* (10 nM), or vehicle (C) for 8 h. mRNA levels were determined for TNFα, IL-6, IL-1β, CCL2, CCL3, and CCL4. (H) BMDMs were pretreated with LPS and then with 0.2–24 nM EI-tPA for 8 h. TNFα mRNA was determined. EI-tPA concentrations of ≥0.5 nM yielded significant results (P < 0.05). (I) BMDMs from Cre-ER^T-negative (control) and Cre-ER^T-positive (Cre-ERT) LRP1^flox/flox mice were treated with TAM (+TAM) or vehicle (−TAM) for 7 d. The Cre-ER^T-negative cells and Cre-ER^T-positive cells that were not treated with TAM both expressed LRP1. The cells were then treated with EI-tPA, α₂M*, or vehicle (C) for 8 h. TNFα mRNA was determined (mean ± SEM; n = 4; **P < 0.01, ***P < 0.001; one-way ANOVA with Dunnett's or Tukey’s post hoc analysis).

Fig. S3.

α₂M* and EI-tPA express antiinflammatory activity by an LRP1-dependent mechanism. (A–E) BMDMs were isolated from wild-type C57BL/6J mice and treated for 8 h with El-tPA (12 nM), α₂M* (10 nM), or vehicle (C). RTqPCR was performed to determine mRNA levels for IL-6, CCL2, CCL3, Arg-1, and TGFβ (mean ± SEM; n = 4; **P < 0.01, ***P < 0.001; one-way ANOVA with Dunnett’s post hoc test). (F–H) LRP1 was deleted in BMDMs by treating cells isolated from Cre-ER^T-positive-LRP1^flox/flox mice with 5 µM TAM (+TAM) for 7 d (solid bars). LRP1-expressing BMDMs included cells isolated from Cre-ER^T-negative-LRP1^flox/flox mice (open bars) and cells isolated from Cre-ER^T-positive-LRP1^flox/flox mice and treated with vehicle (−TAM, shaded bars). The cells were treated with EI-tPA, α₂M*, or vehicle (C) for 8 h. mRNA levels for IL-6, CCL3, and CCL4 were determined by RT-qPCR (mean ± SEM; n = 4).

Fig. S4.

RAP and LF do not further stimulate expression of proinflammatory mediators when LRP1 is deleted. LRP1 was deleted in BMDMs by treating cells isolated from Cre-ER^T-positive-LRP1^flox/flox mice with 5 µM TAM (+TAM) for 7 d (solid bars). LRP1-expressing BMDMs included cells isolated from Cre-ER^T-negative-LRP1^flox/flox mice (open bars) and cells isolated from Cre-ER^T-positive-LRP1^flox/flox mice and treated with vehicle (−TAM, shaded bars). The cells were treated with LF (150 nM), RAP (150 nM), or vehicle (C). RT-qPCR was performed to determine expression of: (A) TNFα; (B) IL-1β; (C) CCL2; (D) CCL3; and (E) CCL4 (mean ± SEM; n = 4).

Fig. 5.

LRP1 regulates NFκB activation. (A) BMDMs from C57BL/6J mice were treated with 150 nM RAP for the indicated times. (B) BMDMs were treated with vehicle (C), LPS (0.1 µg/mL), LF (150 nM), or RAP (150 nM) for 30 min. (C) The same incubations were conducted for 8 h. Immunoblot analysis was performed to detect phospho-IκB and total IκB. (D and E) BMDMs were pretreated with JSH-23 (10 μM), LY294002 (20 μM), or vehicle for 16 h and then with LF, RAP, or vehicle (C) for 8 h. mRNA levels were determined for TNFα and CCL3 (mean ± SEM; n = 6; **P < 0.01, ***P < 0.001; one-way ANOVA with Tukey’s post hoc test). (F) BMDMs were treated with El-tPA (12 nM) or α₂M* (10 nM) together with LPS (0.1 µg/mL) or with LPS or vehicle (C) alone for 1 h. Immunoblot analysis was performed. (G) The same experiment was performed for 8 h.

Fig. S5.

The effect of LRP1 antagonists on cytokine expression require NFкB. BMDMs were isolated from wild-type mice and pretreated for 16 h with JSH-23 (10 μM) (+) or vehicle (−) and then with LF (150 nM), RAP (150 nM), or vehicle (C) for 8 h. RNA was isolated and RT-qPCR was performed to determine mRNA levels for (A) IL-6; (B) IL-1β; (C) CCL2; and (D) CCL4 (mean ± SEM; n = 3; *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA with Tukey’s post hoc analysis).

Fig. 6.

miR-155 sustains the proinflammatory activity of LRP1 antagonists. (A) BMDMs from Cre-ER^T-positive-LRP1^flox/flox mice were treated with TAM (+TAM) or vehicle (−TAM). miR-155, miR-124, miR-9, and miR-223 were determined by qPCR (mean ± SEM; n = 5; ***P < 0.001). (B) ChIP was performed to detect the miR-155 parent gene promoter in association with RNA polymerase II in BMDMs from Cre-ER^T-positive-LRP1^flox/flox mice treated with TAM (+) or vehicle (−). The miR-9 promoter was analyzed as a control. (C) BMDMs from C57BL/6J mice were treated for 8 h with LPS (0.1 µg/mL), LF (150 nM), RAP (150 nM), GST (150 nM), or vehicle (C). miR-155 was determined (n = 5; *P < 0.05, ***P < 0.001). (D) BMDMs were treated with 150 nM RAP for the indicated times. TNFα mRNA and miR-155 were determined. miR-155 also was determined in cells treated with RAP and 10 µM JSH-23 (+JSH-23). (E) BMDMs from C57BL/6J mice were transfected with 10 nM miR-155 inhibitor (Inh) (+) or with miRNA inhibitor negative control (−). After 48 h, the cells were treated with 150 nM RAP (+) or vehicle (−) for 1 h or 8 h. TNFα mRNA was determined (n = 4, ns, not statistically significant; *P < 0.05). (F) BMDMs were pretreated with TNFα-neutralizing antibody (+) or isotype-matched IgG (−) and then with RAP for 0, 30, or 60 min. Immunoblot analysis was performed. (G) BMDMs were treated with RAP (150 nM) or vehicle (C) alone or in the presence of TNFα-neutralizing antibody (1 µg/mL) or isotype-matched IgG for 8 h. miR-155 was determined. (H) BMDMs were treated with LPS (0.1 µg/mL) alone or together with El-tPA (12 nM) or α₂M* (10 nM) for 8 h. miR-155 was determined (n = 5; **P < 0.01, ***P < 0.001).

Fig. S6.

miR-155 sustains the antiinflammatory activity of LRP1 antagonists. BMDMs from C57BL/6J mice were transfected with miR-155 inhibitor (10 nM) (+) or with miRNA inhibitor negative control (−). After 48 h, the cells were treated with 150 nM RAP (+) or with vehicle (−) for 1 h or 8 h. mRNA levels for (A) IL-6 and (B) CCL4 were determined by RT-qPCR (mean ± SEM; n = 4; ***P < 0.001; ns, not statistically significant; one-way ANOVA followed by Tukey’s post hoc analysis).