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

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.


Introduction
IL-1 , released by activated monocytes and macrophages, is a critical player in proinflammatory pathways activation in nearly every tissue and organ [1][2][3]. In particular, IL-1 is implicated in both acute and chronic inflammation and in cell growth, differentiation, angiogenesis, profibrogenic mediators production, immune response regulation, and carcinogenesis [1][2][3][4][5][6][7][8][9][10][11]. A fine-tune control of its secretion is required and achieved by a two-steps mechanism acting both at transcriptional and at posttranslational level. Firstly, the inactive cytoplasmic precursor (pro-IL-1 ) is synthesized; subsequently the active protein is produced by Caspase-1 proteolytic cleavage of the precursor and released into the extracellular space where it initiates its wide range of responses [1][2][3][4][5]12]. The first step of the mechanism requires the activation of specific complex pathways, such as nuclear factor-kB (NF-kB) and extracellular-signal regulated kinases 1/2 (ERK 1/2) [4,6,13]. The second step requires the activation of molecular platforms called inflammasomes, inducing the activation of the proteolytic enzyme Caspase-1. Inflammasomes are composed of nucleotide-binding domain leucine-rich repeat (NLR) receptors, adaptor proteins, and the effector protein pro-Caspase-1 [1][2][3][4][5][6]. The most extensively studied inflammasome is NLRP3/NALP3, formed by the NLR pyrin domain containing 3 (NLRP3, also known as NALP3 or cryopyrin) receptors and by the apoptosis-associated specklike protein containing a Caspase activation and recruitment domain (ASC) adapter [1][2][3][4][5][6]. NALP3 inflammasome activation, induced by a large variety of endogenous and exogenous stimuli, appears to occur in two steps. The first one, priming/licensing step, leads to the activation of 2 Mediators of Inflammation NF-KB-mediated signaling, which in turn upregulates transcription of inflammasome-related components, including inactive NALP3 and pro-IL-1 . The second one is the oligomerization of NALP3 and subsequent assembly of NALP3/ASC/pro-Caspase-1 to a complex triggering the activation of pro-Caspase-1 in Caspase-1 active fragment, as well as the production and secretion of mature IL-1 . Once activated, NALP3 represents a major player in innate immune host defense [2-4, 6, 14].
IL-1 is a strong regulator of Brain Natriuretic Peptide BNP (BNP) [29], a member of the Natriuretic Peptides (NPs) family, synthetized and secreted constitutively by the heart, deeply involved in body fluid and cardiovascular homeostasis [30,31]. BNP is upregulated in myocardium in response to many pathophysiological stimuli and exerts its biological functions by guanylyl cyclase Natriuretic Peptide Receptor-1 (NPR-1/NPR-A) [30][31][32][33][34]. NPR-1 activation induces an increase of cGMP intracellular levels, leading to the activation of specific cGMP-dependent protein kinases, phosphodiesterases and cyclic nucleotide gated cation channels [30,31,34]. NPs and NPR-1 are expressed in many tissues and exert endocrine, autocrine, and paracrine effects in normal or pathological conditions [30,31,34] and a wide range of further activities, including an emerging role on inflammation and immunity [30,31,[35][36][37][38]. In particular, BNP plasma level is upregulated in patients during inflammatory contexts, such as sepsis or septic shock [37,39] and in several inflammatory diseases with or without cardiac dysfunction [37]. In addition, the main cytokines released during inflammation increase BNP production and secretion in vitro [37,40,41] and in many conditions where IL-1 and other cytokines levels are increased [42], BNP plasma levels may be particularly elevated [43]. Finally, BNP inhibits monocytes chemotaxis [35] and influences some inflammatory mediators production in macrophages [36] and NPR-1 is expressed on immune cells [30,35,38,44,45]. Altogether, these observations suggest a role for BNP in inflammation and its possible immunomodulatory action. However, the relationship between BNP/inflammation/immune systems still remains to be widely investigated. In addition, despite BNP modulates some proinflammatory cytokines production [36], no data exist about its effect on human IL-1 secretion.
The aim of our study was to evaluate BNP effect on IL-1 release and NF-kB, ERK 1/2, NALP3, ASC, and Caspase-1 activation in LPS/ATP stimulated human THP-1 monocytes. For the first time to our knowledge, we demonstrated that BNP/NPR-1/cGMP axis strongly downregulated IL-1 secretion from THP-1 monocytes by inhibiting NF-kB and ERK 1/2 activation and all the elements of NALP3 inflammasome cascade. Analyzing the mechanism of action of BNP-dependent NALP3 inflammasome inhibition, we demonstrated that it is directly related to BNP deregulatory effect on NF-kB/ERK 1/2 activation. Our data, demonstrating such multiple inhibitory actions of BNP, reveal a novel potent anti-inflammatory and immunomodulatory role for this peptide. In addition, our study suggests that BNP might be a promising therapeutic agent for the treatment of inflammatory/immune-related and IL-1 /NF-kB/ERK1/2/NALP3/ASC/Caspase-1-associated diseases and, in general, in those pathophysiological scenarios where inflammatory and immune networks are involved.

Cell Culture and Drug Treatments.
Human THP-1 monocytes were purchased from American Type Culture Collection (ATCC, USA) and routinely maintained at 37 ∘ C in 5% CO 2 in RPMI 1640 supplemented with 10% heat inactivated (1 h at 56 ∘ C) FBS, 1x L-glutamine, 1 mM sodium pyruvate, 1x nonessential amino acids, 100 units/mL of penicillin, and 0.1 mg/mL of streptomycin (Invitrogen, Italy). THP-1 cells were plated (2 × 10 6 cells/well) in 35 mm six-well culture dishes and treated for 30 min or 24, 48, and 72 h with 10 g/mL LPS/5 mM ATP in the presence or absence of 10 −6 and 10 −8 M human BNP. In independent experiments, 10 M BAY 11-7082 or U0-126 were added to cells 1 h before LPS/ATP treatment. In independent experiments, THP-1 cells were differentiated to macrophage-like cells by 20 nM PMA for 72 h, washed three times with PBS, and plated (2 × 10 6 cells/well) in 35 mm six-well culture dishes and, after 24 h, treated for 30 min with 10 g/mL LPS/5 mM ATP in the presence or absence of 10 −8 M human BNP. DMSO was used at 0.0005% assay concentration producing no significant toxicity. Control cells with DMSO did not show any significant difference with respect to control cells in RPMI 1640 medium; therefore all the relative treatments were compared to these latter controls.

Cell Viability.
Cell viability was measured with a standard trypan blue uptake assay. Cell cultures were also morphologically examined via light microscopy.

Measurements of Secreted IL-1 .
Measurements of secreted IL-1 were performed in 100 L supernatant after THP-1 exposure to the indicated compounds for 24, 48, and 72 h at 37 ∘ C. After treatments, supernatants were collected and human IL-1 levels were determined by the specific ELISA kit, according to the manufacturer's guidelines (eBioscience, USA).

cGMP
Measurements. cGMP intracellular levels were determined by the specific EIA kit, according to the manufacturer's guidelines (GE Healthcare, UK). THP-1 cells were plated (1 × 10 6 cells/well) in 48-well culture dishes, incubated for 5 and 30 min with 10 −8 M human BNP, in absence or presence of 10 ug/mL LPS/5 mM ATP, and lysed in 200 L of the provided lysis buffer. Measurements were performed in 50 L of the lysate.

Statistical Analysis.
Results were expressed as means ± SD of three independent experiments performed in triplicate. The statistical significance of differences between treated and untreated cells was assessed by Student's -test. Differences between groups were considered significant when < 0.05. , and 1(f)), thus suggesting an intense NALP3 inflammasome activation.

NF-kB and ERK 1/2 Are Involved in NALP3/ASC/Caspase-1 Cascade Activation in THP-1 Cells.
Both NF-kB and ERK 1/2 can be involved in NALP3 and Caspase-1 activation [4,6,46]. We then demonstrated the effective involvement of these pathways in our system, using the specific inhibitors

BNP Is Involved in All the Molecular Mechanisms Controlling IL-1 Production and Release by Inhibiting the Activation of NF-kB, ERK 1/2, and NALP3/ASC/Caspase-1 Cascade in
Macrophage-Like Cells. BNP effect on the molecular mechanisms controlling IL-1 production and release was finally evaluated also in macrophages. By using PMA differentiated THP-1 cells, we demonstrated that 10 −8 M BNP treatment abrogated and downregulated 30 min LPS/ATP-induced NF-kB and ERK 1/2 activation, respectively (Figure 6(a)). Regarding BNP effect on NALP3 cascade, we demonstrated that 10 −8 M BNP treatment abrogated 30 min LPS/ATPinduced NALP3 protein expression and Caspase-1 activation and downregulated ASC protein expression, respectively ( Figure 6(b)). Altogether these results demonstrate BNP inhibitory action on all the steps of the mechanism controlling IL-1 production and secretion also in macrophage-like cells.

Discussion
IL-1 is a central mediator in inflammation and immunity. IL-1 production and release are tightly regulated by the activation of complex pathways such as NF-kB and ERK1/2, to induce pro-IL-1 upregulation, and NALP3/ASC/Caspase-1, to cleave pro-IL-1 into the biologically active secreted protein [4,6,12,13,46]. Considering the pathogenic roles of IL-1 , NF-kB, ERK1/2, and NALP3 inflammasome inappropriate activation in a large number of major human disorders and in cancer [1,2,[4][5][6][7][8][9][10][11][15][16][17][18][19][20][21][22][23][24][25][26][27][28], a large interest is focused on the identification of molecules able to target them, in order to develop new therapeutic agents. IL-1 is a potent regulator of BNP [29], a hormone/paracrine/autocrine factor mainly involved in body fluid and cardiovascular homeostasis, via the guanylyl cyclase NPR-1 [30,31,34]. An emerging role for BNP in inflammation and immune system has been suggested, even though it remains largely unexplored. Here, we newly demonstrated that, in human THP-1 monocytes, LPS/ATP-induced IL-1 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 cascade (Figure 7). Due to the pivotal role of IL-1 on inflammation and immunomodulation, finding that an endogenous molecule, such as BNP, is able to strongly downregulate its release  Figure 6: BNP is involved all the molecular mechanisms controlling IL-1 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 −8 M BNP in absence or presence of 10 g/mL LPS/5 mM ATP for 30 min. Cell lysates were immunoblotted for P-IkB-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--actin, to confirm equal loading. Western blots are representative of three separate experiments.
is particularly important. Moreover, these data support the anti-inflammatory and immunomodulatory role of BNP on human monocytes.
Our results, in line with BNP modulatory effect on other inflammatory mediators [36], suggest that during inflammation, where a complex network of pleiotropic mediators from plasma or cells (including monocytes and macrophages) occurs, BNP, by inhibiting IL-1 secretion, can affect the injured tissues-related inflammatory reactions, contributing to restoring cytokines production balance. In fact, since IL-1 stimulates the production and release of additional cytokines, BNP, by inhibiting it, can play an important compensatory role. Besides, in conditions where IL-1 and other cytokines levels are increased, such as after myocardial infraction and during progression of heart failure [42], elevated BNP plasma levels have been found [43]. Moreover, BNP levels increase also in patients during inflammatory contexts such as severe sepsis or septic shock [37,39], and in several inflammatory pathologies with or without cardiac dysfunction [37]. Since IL-1 is a strong regulator of BNP promoter [29], we suggest that the observed BNP increase during IL-1 -related inflammatory contexts might be due to the direct effect of this cytokine. Concomitantly, the here found BNP inhibition of IL-1 release might contribute also to balance and/or restore BNP secretion itself, by an autocrine/paracrine loop. In support, NPR-1 is expressed on human monocytes [35,38], macrophages [44], monocyte-derived dendritic cells [45], and peripheral blood mononuclear cells [35]. In agreement, here we newly report that THP-1 cells respond to BNP with an increase in cGMP production, thus indicating NPR-1 activation. Therefore, we suggest that in human monocytes, by inhibiting IL-1 , the BNP/NPR-1/cGMP axis has an immune-regulatory and anti-inflammatory role. In addition, we suggest that BNP might exert these actions also in tissue microenvironment, where BNP autocrine/paracrine mechanisms may occur [31]. This appears to be of special interest also in the pathogenesis of cardiovascular diseases, including hypertrophy, myocardial infraction, and heart failure, where the inflammatory response takes place and BNP release is particularly elevated [31][32][33]. Analyzing the molecular mechanism of BNP inhibitory action on IL-1 secretion, we firstly demonstrated the involvement of NF-kB and ERK1/2 pathways in our system, their inhibitors being able to strongly downregulate LPS/ATP-induced pro-and IL-1 production. Subsequently, we demonstrated BNP's capability to inhibit both these pathways and pro-IL-1 protein expression. The observed BNP inhibitory action on NF-kB and ERK 1/2 activation in human monocytes has been confirmed also in macrophages-like cells, in agreement with similar results obtained in animal models [47,48]. Regarding NF-kB, we demonstrated that BNP inhibited IkB-phosphorylation, essential for the release of the active form of this transcription factor [15]. Even though we did not investigate BNP inhibitory mechanism on NF-kB and ERK 1/2 activation, we hypothesize that this might be related to multiple processes. For instance, BNP inhibition of IkB-phosphorylation could be the result of an indirect mechanism, occurring via BNPinduced ERK1/2 downregulation, this latter being a pathway involved in NF-kB activation by IkB-phosphorylation [18]. Interestingly, the here observed BNP inhibition of IkBphosphorylation could be implicated also in the inhibitory effect of BNP on ERK 1/2, being IKK family involved in both NF-kB and ERK1/2 activation [17]. In addition, BNP-induced ERK 1/2 inhibition might be ascribed also to the here found BNP-induced increase in cGMP levels. In fact, both increased cGMP levels [49] or cGMP-dependent kinases exert a direct downregulation of ERK 1/2 activation [50]. In addition, we do not exclude that both NF-kB or ERK 1/2 inhibitions may be the result of a negative feed-back loop via IL-1 , this cytokine being an activator of both these pathways [1,15]. Finally, such inhibitions could be related to the here observed downregulatory effect of BNP on ASC expression, ASC itself being an important activator of both these pathways [2,28]. The biological relevance of BNP-induced NF-kB/ERK1/2 inhibitions is that BNP may exert an anti-inflammatory and immunomodulatory role via the regulation of such pathways. In fact, both these signaling are involved in LPS-induced proinflammatory responses and their activation result in a rapid release of proinflammatory cytokines into the circulation, leading to an additional inflammatory response [15,46]. In addition, it has been demonstrated in mouse macrophages that NF-kB activation additionally turns on NALP3 transcription, thereby licensing NALP3 inflammasome activation [6,51]. Therefore, we suggest that BNP inhibitory effect on NF-kB and ERK 1/2 can be further investigated for a potential therapeutic employment of this hormone in the treatment of inflammatory diseases, where the application of chemical NF-kB and ERK 1/2 inhibitors may cause problems related to their efficacy and side effects [52,53]. Besides, BNP infusion is used on top of standard care, providing clinical benefit in acutely decompensated heart failure patients [54,55].
Regarding the second part of the mechanism controlling IL-1 secretion, we showed that BNP completely reverted LPS/ATP-induced NALP3, ASC and pro-Caspase-1 protein expression and NALP3 inflammasome activation both in monocytes and in macrophage-like cells. BNP capability to downregulate NALP3/ASC/Caspase-1 protein expression demonstrates its involvement in the priming/licensing step of inflammasome activation in these different cellular systems and represents an important new mechanism of action for this natriuretic peptide. In addition, we demonstrated that BNP completely blocked LPS/ATP-induced Caspase-1 active fragment production, both in THP-1 monocyte and in macrophage-like cells. Since the hallmark of NALP3 inflammasome activation is the proteolytic cleavage of Caspase-1 that, in turn, cleaves pro-IL-1 into the mature form IL-1 , our results indicate BNP involvement also in the second mechanism of inflammasome activation. This effect appears to be particularly important since NALP3 inflammasome components may have roles also in inflammasomeindependent cytokine production and noninflammasomerelated cytokines can influence the activation of the inflammasome [6]. Therefore, we suggest that BNP may exert anti-inflammatory and immunomodulatory effect also via the downregulation of both the elements of this molecular platform and its activation, implicated in the pathogenesis of major human diseases with a high prevalence worldwide [1,[4][5][6][7][8][9][10][11][19][20][21][22][23][24][25][26][27]. Hence, BNP could be further investigated also for its potential therapeutic effect on NALP3/ASC/Caspase-1 associated disorders also in consideration of the fact that many of the autoimmune diseases are refractory to most therapies and that immunosuppressive agents have many side effects [6]. Analyzing the mechanism of action of BNP inhibitory effect on NALP3/ASC/Caspase-1 cascade, we finally demonstrated that it is directly related to BNP deregulatory effect on NF-kB and ERK 1/2 activation. In fact, the specific BAY 11-7082 and U0-126 inhibitors completely reverted LPS/ATP-induced NALP3, ASC, and Caspase-1 protein expression and Caspase-1 activation. These results are in agreement with previous data demonstrating that NALP3 expression is dependent on NF-kB activation [4,51] and that BAY 11-7082 is able to inhibit both NALP3 inflammasome and Caspase-1 activation in a murine model [56]. Moreover, it has been recently demonstrated that NALP3 inflammasome activation by LPS requires ERK 1/2 and that U0-126 significantly blocks it [46].

Conclusions
In conclusion, we demonstrated a novel role for BNP/NPR-1/cGMP axis: downregulation of IL-1 secretion from THP-1 monocytes, via a strong inhibition of all the molecular mechanisms controlling its production and secretion: NF-kB, ERK 1/2, and NALP3/ASC/Caspase-1 cascade. Regarding BNP inhibitory effect on NALP3 inflammasome activation, we demonstrated that it is related to BNP-induced downregulation of NF-kB and ERK 1/2 activation. Our data point out a potent anti-inflammatory and immunomodulatory role for this peptide suggesting a possible employment of this endogenous agent, already used for the treatment of heart failure [54,55], also in the treatment of inflammatory/immune-related and IL-1 /NF-kB/ ERK1/2/NALP3/ASC/Caspase-1-associated diseases, known to affect millions of people worldwide.