A Novel Role for Brain Natriuretic Peptide: Inhibition of IL-1β Secretion via Downregulation of NF-kB/Erk 1/2 and NALP3/ASC/Caspase-1 Activation in Human THP-1 Monocyte

Abstract

Interleukin-1β (IL-1β) is a pleiotropic cytokine and a crucial mediator of inflammatory and immune responses. IL-1β processing and release are tightly controlled by complex pathways such as NF-kB/ERK1/2, to produce pro-IL-1β, and NALP3/ASC/Caspase-1 inflammasome, to produce the active secreted protein. Dysregulation of both IL-1β and its related pathways is involved in inflammatory/autoimmune disorders and in a wide range of other diseases. Identifying molecules modulating their expression is a crucial need to develop new therapeutic agents. IL-1β is a strong regulator of Brain Natriuretic Peptide (BNP), a hormone involved in cardiovascular homeostasis by guanylyl cyclase Natriuretic Peptide Receptor (NPR-1). An emerging role of BNP in inflammation and immunity, although proposed, remains largely unexplored. Here, we newly demonstrated that, in human THP-1 monocytes, LPS/ATP-induced IL-1β secretion is strongly inhibited by BNP/NPR-1/cGMP axis at all the molecular mechanisms that tightly control its production and release, NF-kB, ERK 1/2, and all the elements of NALP3/ASC/Caspase-1 inflammasome cascade, and that NALP3 inflammasome inhibition is directly related to BNP deregulatory effect on NF-kB/ERK 1/2 activation. Our findings reveal a novel potent anti-inflammatory and immunomodulatory role for BNP and open new alleys of investigation for a possible employment of this endogenous agent in the treatment of inflammatory/immune-related and IL-1β/NF-kB/ERK1/2/NALP3/ASC/Caspase-1-associated diseases.

Figure 1

BNP decreases LPS/ATP-induced IL-1β release in THP-1 cells. THP-1 cells were treated with 10⁻⁶ and 10⁻⁸M BNP in absence or presence of 10 μg/mL LPS/5 mM ATP for 24 (a, d), 48 (b, e), and 72 h (c, f). The supernatants were collected and IL-1β release was measured by ELISA (a, b, c). Cell viability was assessed by trypan blue uptake assay (d, e, f). Histograms indicate means ± SD of three separate experiments each one tested in triplicate. ^∗∗∗P < 0.001 versus untreated cells and °P < 0.05, °°P < 0.01, and °°°P < 0.001 versus LPS/ATP treated cells.

Figure 2

BNP activates NPR-1 in THP-1 cells. (a) THP-1 cells were treated with 10⁻⁸M BNP in absence or presence of 10 μg/mL LPS/5 mM ATP and cGMP intracellular levels were evaluated after 5 and 30 min treatment. (b) THP-1 cells were treated with 10⁻⁸M BNP in absence or presence of 10 μg/mL LPS/5 mM ATP for 30 min. Cell lysates were immunoblotted for NPR-1. The blots were stripped of the bound Ab and reprobed with mouse anti-β-actin, to confirm equal loading. Western blots are representative of three separate experiments. Histograms indicate means ± SD of three separate experiments each one tested in triplicate. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 versus untreated cells.

Figure 3

BNP is involved in the priming mechanism controlling IL-1β production by inhibiting NF-kB and ERK 1/2 activation in THP-1 cells. (a) THP-1 cells were incubated with 10 μM BAY 11-7082 or U0-126 for 1 h and then treated with 10 μg/mL LPS/5 mM ATP for 48 h. The supernatants were collected and IL-1β release was measured by ELISA. (b, c, d) THP-1 cells were treated with 10⁻⁸M BNP in absence or presence of 10 μg/mL LPS/5 mM ATP for 30 min (b, c) or 48 h (b, c, d). Cell lysates were immunoblotted for P-IkB-α (b), P-ERK 1/2 (c), or pro-IL-1β (d). (e, f) THP-1 cells were incubated with 10 μM BAY 11-7082 (e) or U0-126 (f) for 1 h and then treated with 10 μg/mL LPS/5 mM ATP for 48 h. Cell lysates were immunoblotted for pro-IL-1β. The blots were stripped of the bound Ab and reprobed with mouse anti-β-actin, to confirm equal loading. Western blots are representative of three separate experiments. All histograms indicate means ± SD of three separate experiments each one tested in triplicate. ^∗∗P < 0.01 and ^∗∗∗P < 0.001 versus untreated cells and °P < 0.05, °°P < 0.01, and °°°P < 0.001 versus LPS/ATP treated cells.

Figure 4

BNP is involved in the second mechanism controlling IL-1β production by inhibiting NALP3, ASC, and Caspase-1 cascade activation in THP-1 cells. THP-1 cells were treated with 10⁻⁸M BNP in absence or presence of 10 μg/mL LPS/5 mM ATP for 30 min. Cell lysates were immunoblotted for NALP3 (a), ASC (b), or Caspase-1 (c). The blots were stripped of the bound Ab and reprobed with mouse anti-β-actin, to confirm equal loading. Western blots are representative of three separate experiments. Histograms indicate means ± SD of three separate experiments each one tested in triplicate. ^∗P < 0.05 and ^∗∗P < 0.01 versus untreated cells and °°P < 0.01 and °°°P < 0.001 versus LPS/ATP treated cells.

Figure 5

NF-kB and ERK 1/2 are involved in NALP3/ASC/Caspase-1 cascade activation in THP-1 cells. THP-1 cells were incubated with 10 μM BAY 11-7082 (a, b, c) or U0-126 (d, e, f) for 1 h and then treated with 10 μg/mL LPS/5 mM ATP for 30 min. Cell lysates were immunoblotted for NALP3 (a, d), ASC (b, e), or Caspase-1 (c, f). The blots were stripped of the bound Ab and reprobed with mouse anti-β-actin, to confirm equal loading. Western blots are representative of three separate experiments. Histograms indicate means ± SD of three separate experiments each one tested in triplicate. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 versus untreated cells and °P < 0.05, °°P < 0.01, and °°°P < 0.001 versus LPS/ATP treated cells.

Figure 6

BNP is involved all the molecular mechanisms controlling IL-1β production and release by inhibiting the activation of NF-kB, ERK 1/2 and of all the elements of NALP3 inflammasome/ASC/Caspase-1 cascade in macrophage-like cells. PMA differentiated THP-1 cells were treated with 10⁻⁸M BNP in absence or presence of 10 μg/mL LPS/5 mM ATP for 30 min. Cell lysates were immunoblotted for P-IkB-α and P-ERK1/2 (a) or NALP3, ASC, or Caspase-1 (b). The blots were stripped of the bound Ab and reprobed with mouse anti-β-actin, to confirm equal loading. Western blots are representative of three separate experiments.

Figure 7

Mechanism of action of BNP inhibitory effect on LPS/ATP-induced IL-1β secretion in human THP-1 monocyte. BNP/NPR1/cGMP axis strongly inhibits IL-1β secretion by acting both at transcriptional and at posttranslational level. Firstly, by inhibiting the activation of NF-kB and ERK 1/2 signaling pathway, BNP downregulates the production of the inactive cytoplasmic precursor pro-IL-1β. Subsequently, again by inhibiting NF-kB and ERK 1/2 activation, BNP strongly downregulates all the elements of NALP3 inflammasome/ASC/Caspase-1 cascade and Caspase -1 activation, essential steps for the formation of the active secreted IL-1β protein.